Lateral flow assays using two dimensional features

ABSTRACT

The present invention relates to novel lateral flow devices using two dimensional features, preferably, uniform two dimensional test and control features, and the methods for detecting an analyte using the lateral flow devices, and processes for making the lateral flow devices.

I. CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. provisionalapplication Ser. No. 61/461,499, filed Jan. 18, 2011, the content ofwhich is incorporated by reference in its entirety.

II. TECHNICAL FIELD

The present invention relates to novel lateral flow devices using twodimensional features, preferably, uniform two dimensional test andcontrol features, and the methods for detecting an analyte using thelateral flow devices, and processes for making the lateral flow devices.

III. BACKGROUND OF THE INVENTION

Lateral flow assays dominate the non-glucose rapid testing market inhumans, and also other areas of application that require rapidgeneration of a test result, including veterinary diagnostics,agricultural testing, bio-warfare testing and food safety testing, asexamples.

The advantages of the lateral flow assay system (LFIA) are well known(see Table 1). Critical among these advantages, is that they representan appropriate point of care and field use technology that can bebrought to market quickly and for a relatively small investment, and beapplied over a very broad range of applications.

TABLE 1 Benefits of Lateral Flow Assays in Point of Need ApplicationsKnown and mature technology Relative ease of manufacture—equipment andprocesses already developed and available Easily scalable to high volumeproduction Stable—shelf lives of 12-24 months often withoutrefrigeration Ease of use: Minimal operator dependent steps andinterpretation Can handle small volumes of multiple sample types Can beintegrated with onboard electronics, reader systems and informa- tionsystems Can have high sensitivity, specificity, good stabilityRelatively low cost and short timeline for development and approvalMarket presence and acceptance—minimal education required for users andregulators

Traditionally designed lateral flow assays suffer from performancelimitations, most notably low sensitivity and poor reproducibility.Additionally, standard format lateral flow assays produce results in theform of the presence or absence of lines at test and control regions,which have to be interpreted by eye. This subjective interpretation canoften be difficult and can lead to incorrect results. These limitationshave been exacerbated by the continuing use of traditional developmentand manufacturing practices, materials, labels, and visual detectionsystems. The reliance on the generation of linear features such ascontinuous lines to indicate the presence or absence of analyte in thesystem is a function of a variety of mechanical factors that can inhibitthe reproducible formation of other features on the same substrate. Inmany instances it is desirable to test for multiple reactions in asingle sample (multiplexing). The use of linear features makesmultiplexing difficult, makes interpretation of competitive assayresults counter-intuitive, and adds to result variability through usererror.

Further, quantification and objective read/record technology, oftenlinked to laboratory information systems (LIS) are being implemented atan increasing rate. Demand for multiplexed systems, where detection ofmore than one analyte is necessary in the same test system is alsodeveloping. Current Lateral Flow test systems are generally incompatiblewith these needs.

The limitations of the current lateral flow assay devices and methodshave substantially restricted their application to relatively lowspecification testing. For more demanding systems requiring highsensitivity, quantification and multiplexing, and for numerous marketsegments such as military, consumer, environmental or veterinary testingwhere intuitive, fast, easily interpreted results are required, thecurrent Lateral Flow system is inadequate.

Therefore, there is a need for devices and methods that overcome thelimitations of the current technologies and methodologies, that are lesssubject to interpretation errors, that can produce quantitative results,that are reproducible, that can be multiplexed and that can be appliedin numerous market segments. The present invention addresses these andother related needs.

The invention disclosed herein provides for such a device, and disclosesmethods including but not limited to, methods for manufacturing rapidassays that can produce easily interpreted results in the form of uniqueindicia, which indicia being representative of the results of the test.The indicia may be standardized symbols visible to the eye or a readeror may be encrypted indicia interpretable by a specialized readerdevice. The indicia can be developed in any orientation relative to thedirection of flow of the assay. The indicia may also indicatequalitative outcomes (positive or negative), semi-quantitative orquantitative outcomes depending upon the assay and reagent design.

Lateral Flow Assay Formats

FIG. 1 shows a typical configuration for such a lateral flow assay.Traditionally designed assays are composed of a variety of materials,each serving one or more purposes, overlapping onto one another, mountedon a backing card using a pressure sensitive adhesive.

The test device consists of several zones, typically constituted byindividual segments of different materials, each of which will bebriefly explained here. When a test is run, a sample of the material tobe tested (sample) is added to the proximal end of the strip, onto aSample Application Pad. Here, the sample is treated by means of addedpredetermined reagents to make it compatible with the rest of the test.Liquid phase elements of the treated sample (which may be dissolved,suspended, emulsified or any other liquidized formats) migrate to a nextsegment of the test device, the Conjugate Pad. Here, a detector reagenthas been immobilized, typically consisting of a protein linked passivelyor covalently to a signal molecule or particle, typically a colloidalgold, or a colored, fluorescent or paramagnetic monodisperse latexparticle. The signal reagent can also be another reagent, includingnon-particulates (e.g., soluble, directly labeled fluorophores gels).This label has been conjugated to one of the specific biologicalcomponents of the assay, either an antigen or an antibody, depending onthe assay format of the specific test device. The liquid phase samplere-mobilizes the dried conjugate material causing it to incorporate intothe liquid phase sample material, and analyte in the sample interactswith the conjugate as both migrate into the next section of the teststrip, the Reaction Matrix. The reaction matrix is typically a porousmembrane with a hydrophilic, open structure for the purposes oftransporting liquids to the reagent and control, onto which the otherspecific biological components of the assay have been immobilized. Theseare typically proteins, either antibody or antigen, which have been laiddown in bands or stripes in specific areas of the membrane where theyserve to capture the components of the liquid phase sample, the analyteand conjugate, as they migrate past, through or over the capture lines.Excess liquid phase materials (sample and reagents) continue to migrateacross the strip, past the capture lines and are entrapped in a Wick orabsorbent pad. Test results are developed on the reaction matrix and arerepresented as the presence of absence of indicia (typically continuouslines) of captured conjugate which are read either by eye or using areader device.

Assay formats are often either sandwich (direct) or competitive(competitive inhibition) in nature, and can accommodate qualitative,semi-quantitative, or in certain specific cases, fully quantitativeassays.

Direct assay formats are typically used when testing for larger analyteswith multiple antigenic sites, such as hCG, Dengue antibody or antigen,or HIV. In this case, a positive result is indicated by the presence ofa test line. Some of the conjugated particles will not be captured atthe capture line, and will continue to flow toward the second line ofimmobilized antibodies, the control line. This control line typicallycomprises a species-specific anti-immunoglobulin antibody, specific forthe conjugate antibody on the conjugate.

Competitive assay formats are typically used when performing a test forsmall molecules with single antigenic determinants, which cannot bind totwo antibodies simultaneously. In this format, a positive result isindicated by the absence of a test line on the reaction matrix. Acontrol line should still form, irrespective of the result on the testline. The two formats are illustrated schematically in FIGS. 2 a and 2b.

Fluid Transport and Signal Development in Lateral Flow Systems

The function of the current lateral flow test device is based oncapillary flow of liquids along the length of the test strip, flowingfrom the sample introduction pad to the absorbent pad as shown inFIG. 1. Hence the flow geometry and capillary driving force isessentially one dimensional through the reaction matrix, and through thetest and control lines. Nitrocellulose membranes are the predominantlyused reaction matrix in lateral flow tests. In a lateral flow device thetest and control lines are typically made up of proteins but can beother types of biomarker that are bound to the Reaction Matrix in lineformats, generally oriented perpendicular to the direction of flow.

An example describing the processing and use of nitrocellulose as thereaction matrix will illustrate the issues with this line format.

Purpose: The purpose of the reaction matrix in a lateral flow assay isto bind proteins or other capture reagents at the test and controlareas, and to maintain their stability and activity over the shelf lifeof the product. When the test is run, this matrix must accept theconjugate and sample from the conjugate pad, flow them consistently tothe reaction area, allow the reaction at the test and control lines tohappen and allow excess fluids, label and reactants to exit withoutbinding.

Material: The material of choice in the vast majority of lateral flowassay systems has historically been nitrocellulose. Several attemptshave been made to introduce other material types into the market,including nylon and PVDF membranes, however those attempts have hadlimited success, apparently due to factors including cost, limitedutility, the need for education regarding new chemistry and processingrequirements, and inertia due to the large bank of existing experiencein the use of nitrocellulose. Other matrices are in development,including plastic materials with controlled contact angles that allowthe flow of reactants to occur on the surface of the matrix in acontrolled manner.

Nitrocellulose, while extremely functional, may not always be an idealmatrix for an analytical membrane in LFIA's. It does have certaincharacteristics that make it useful, and it remains the only materialthat has been successfully and widely applied in this way to date. Thesecharacteristics include relatively low cost, true capillary flowcharacteristics, high protein binding capacity, relative ease ofhandling (with direct cast, or backed membranes) and a variety ofavailable products with varying wicking rates and surfactant contents.However, the material also possesses a variety of characteristics thatmake it imperfect for this application. These include imperfectreproducibility of performance within and between lots, shelf lifeissues, flammability (primarily in unbacked membranes), variablecharacteristics due to environmental conditions, such as relativehumidity, and being subject to breakage (if unbacked), compression andscoring during processing.

As a result of these issues with the material, developers andmanufacturers spend a considerable amount of time and effort inoptimizing chemistries that overcome some of the inherent materialissues, and in developing manufacturing processes that guaranteeadequate performance over the entire shelf life of the product. Carefulcontrol of the key processes of dispensing, dipping and drying, andattention to chemical and biological treatment of the membrane in orderto prevent the introduction of additional variation into the finishedproduct are critical to success.

Flow Characteristics: In order to function as the reaction matrix in alateral flow system, the material is typically hydrophilic and hasconsistent flow characteristics. Nitrocellulose as a base material ishydrophobic, and is made hydrophilic by the addition of rewetting agentsduring the membrane production process. These rewetting agents aresurfactants, and the amount and type of surfactant, and the surfactantaddition methods differ from manufacturer to manufacturer and also frombrand to brand within a manufacturer. The amount and type of surfactantin the membrane can affect the performance of the assay initially andover time. Not every protein will be compatible with every surfactant.This is one reason for the requirement for screening of multiplemembrane types during development. Nitrocellulose membranes' flowcharacteristics change over time, primarily due to desiccation of themembranes upon storage. Nitrocellulose membranes can be envisaged as asponge, with the pores of the sponge being held open by water. If thatwater is removed, the pores collapse, disrupting the ability of themembrane to wick fluids through it. This results in changes andinconsistencies in flowrate over time. As a result, assays based onnitrocellulose can change their performance characteristics over time,as speed directly affects assay sensitivity, and extended run times canresult in false positive issues. This is a major contributor to thevariability in lateral flow assays.

Critical to the appropriate performance of a lateral flow system is therequirement that the system bind reactants only at the desiredlocations, namely the test and control lines. The protein bindingcapacity of a membrane, its interactions with proteins, and the kineticsof the protein binding process are parameters which will determine howone can apply a given set of proteins onto the membrane and howsensitive the resulting diagnostic test will be. Proteins bind tonitrocellulose through a combination of electrostatic, hydrogen andhydrophobic binding. Consistent and reproducible immobilization ofimmunologically active proteins to test and control lines in lateralflow or flow through assays is one of the key elements to the productionof sensitive, reproducible assays.

Membrane Processing: Nitrocellulose must undergo several processesbefore integration into the final device, those typically beingdeposition of test and control line proteins using quantitativedispensers, drying, typically using forced air ovens at elevatedtemperature, and immersion processes for blocking. To lay down the testand control line proteins, the membrane is striped with protein usingeither contact or non-contact dispensing systems, and is typicallyblocked thereafter to control and stabilize flowrates and hydrationcharacteristics, and prevent non-specific binding. The dispensing methodused for the test and control lines must be as quantitative as possible,and should not be subject to variation due to variations in the materialhydration or absorption characteristics. Non-contact dispensing methodsprovide the best solution for quantitatively dispensing proteins ontonitrocellulose. The purpose of blocking a nitrocellulose membrane is toprevent binding of proteins and labeled conjugate to the membrane atareas other than the test and control lines, where it can bespecifically bound. Blocking also serves other functions, includingmaintenance of hydration of membranes, modification of wicking rates andstabilization of test and control line proteins. Blocking is typicallyperformed by immersion of the membranes in a solution containingproteins, surfactants and polymers, and is a relatively uncontrolledprocess. The blocking method must be carefully optimized and controlledto produce optimal performance in the final product over the entireshelf life of the product. Drying is subsequently performed typically bya combination of blocking to remove surface fluids and forced air atelevated temperatures. Again, this drying process must be carefullyoptimized and controlled to minimize variation in the final product.

This process results in the creation of one or more bound lines ofcapture reagent across the width of the reaction matrix. When the assayis run, the combination of the bound protein and subsequent formation ofa sandwich when reacting with the flowing sample/conjugate increase theflow resistance within the test and control line regions. Resistance toflow can also be increased by the fact that the surfactant in the linehas been to some degree driven away from that region by the dispensingprocess, resulting in a line across the membrane that is morehydrophobic than the areas before and after it, in terms of fluid flowthrough the matrix.

As a result, in a standard lateral flow configuration, the test andcontrol features perturb the flow of fluid and analyte within thesystem. One of the primary reasons for the use of test and control linesthat span the entire width of the device is to ensure that theperturbation is even across the width of the device and that flow in thelongitudinal direction remains even and effectively one-dimensional.This prevents the formation of other more preferred test interpretationfeatures, such as alpha-numeric symbols or quantitative indicia.

Current lateral Flow devices and their associated manufacturingprocesses impose various limitations on their use, accuracy andreproducibility.

1. Multiplexing is difficult.

a. There is a growing requirement in point of need diagnostics for thegeneration of assays that can detect more than one analyte in a singledevice. In a standard configuration, this means dispensing multiplelines perpendicular to the flow direction, separated by distances of 1or more millimeters. A typical issue seen in multiplexed assays of thisnature is “line bleed” where signal generated on one line can “bleed”into the next line, where the conjugate is physically restricted,resulting in the formation of background in the device, which lowers thesensitivity of the assay, and can result in false positives.

b. The dynamics of each assay in the system are different from each ofthe others. Lateral flow assays are extremely time sensitive assays. Thereaction begins as soon as the sample and conjugate mix in the conjugateor sample pad, and continues during migration through the device to thetest and control lines. The reaction at the test line occurs quickly,typically in less than 30 seconds. The flow rate of the reactantsthrough the device can be extremely important to the performance of theassay. Flow rate through an analytical membrane, typicallynitrocellulose, decreases in a non linear fashion with distance from theorigin. As a result, the time taken for the first reaction to reach thefirst capture line in a multi line assay, and that taken for thereaction to reach the last line, can be significantly different. Thishas implications for the ability to generate quantitative assays inmultiplexed formats.

2. Antibody selection must focus on antibodies with extremely highaffinity and “on-rate” (K_(on)). This is due to the fact that thereaction at the test line must occur within only a few seconds. Thismakes antibody selection difficult, and means that laborious selectionmethods, such as dot blots or lateral flow formats must be used, asagainst more ergonomic methods such as ELISA, which may select forantibodies with different binding characteristics. This high affinitymakes it impossible to evenly develop large diameter features in thedirection of flow (including lines, which may show gradation of strengthin the direction of flow). The use of smaller features (“pixels”)combined appropriately into larger features overcomes this issue.

3. Only a single format of result is generated (a horizontal line). Theformation of letters, symbols and lines in any orientation other thanperpendicular to the direction of flow in a lateral flow assay is madedifficult by the dynamics of flow and conjugate binding in the strip.Two simple examples of the difficulty of generating alternative shapesin a lateral flow system are illustrated in FIGS. 3 and 4 (dot and +).

a. Dot: If binding reagent is dotted onto a membrane, reagent flow andbinding characteristics of the binding reagent will result typically inone of three outcomes as shown in FIG. 3: (a) formation of a half moonshape, indicating that conjugate is bound at the leading edge of the dotand the rest of the dot shape does not fill. This indicates acombination of high affinity binding at the leading edge which impedesfurther flow through the dot, with the remainder of the reagent findingthe path of least resistance around the dot; (b) a filled out butgenerally inconsistent dot, indicating a low affinity binding reagent,which is non optimal for the lateral flow format; and (c) no binding,indicating non specificity of the binding reagent or a negative sample.

b. Plus/Minus: A typical embodiment of this format is one where thecontrol line would show up as the minus and the combined test andcontrol would show up as a plus as shown in FIG. 4. In FIG. 4 is shownthe actual development of the plus with the test line dispensed parallelto the 1 dimensional flow. In this case the end of test line closest tothe flow introduction shows the highest level of development by theconjugate while the other end shows development only along the edges ofthe test line. In this case the conjugate does not flow up to theinterior of the line due to the high internal flow resistance. The sameresult would be expected if test and control line positions were swappedin the assay.

4. Interpretation of lines is difficult, particularly in systems thatrely on the eye of the user for interpretation. Additionally, lateralflow assays are typically on the order of 2-8 mm wide. The typicalanalytical membrane is nitrocellulose, which is an inhomogeneousmaterial that is inconsistent both within and between lots. As a result,flow effects are commonly seen that lead to the generation ofinconsistent lines across the width of the device. This inhomogeneity inline development can also be created by process-related factors,including poor lamination or cutting. This uneven line development leadsto further interpretation issues, and can be particularly difficult forreader systems.

The dominant effect of one dimensional flow is shown schematically inFIGS. 5( a), (b) and (c), where a flow resistance in the form of a spotof dispensed protein is placed in the flow path before the test andcontrol lines using dispensed drops of different volumes in differentpositions relative to the line. Development of the test line isperturbed directly in line with the placement of the protein spot.Lateral diffusion does occur in the system but can result in evendevelopment of the feature that the fluid reaches after the spot only ifthe distance between the two features is sufficient. The requireddistance is dependent on the diameter of the spot and the pore size ofthe membrane, and is generally of a distance that makes the formation ofinterpretable alpha numeric or other symbols impossible within theworking dimensions of a test.

IV. DISCLOSURE OF THE INVENTION

In one aspect, the present disclosure provides for a test device fordetecting an analyte in a liquid sample, which device comprises aplurality of reagent dots on a matrix, wherein at least two of saidreagent dots do not overlap and are sufficiently spaced apart from eachother so that when said liquid sample flows laterally along said matrix,flow of said liquid sample to, through and/or around one of said tworeagent dots does not substantially affect flow of said liquid sampleto, through and/or around said other reagent dot, each of said tworeagent dots is neither a reagent line across the entire width of saidmatrix in a direction perpendicular to the direction of said liquidsample flow nor a complete circle of a reagent line, and after a liquidsample flows laterally along said test device and passes said at leasttwo reagent dots, said at least two reagent dots form a predeterminedpattern to indicate presence, absence and/or amount of said analyte insaid liquid sample.

In another aspect, the present disclosure provides for a method fordetecting an analyte using the above test device. In one exemplaryembodiment, the present disclosure provides a method for detecting ananalyte in a liquid sample, which method comprises a) contacting aliquid sample with the above test device, wherein the liquid sample isapplied to a site of the test device upstream of the at least two of thereagent dots; b) transporting an analyte, if present in the liquidsample, to the at least two of the reagent dots; and c) assessing thepresence, absence, amount and/or pattern of signal(s) generated at theat least two of the reagent dots to determining the presence, absenceand/or amount of the analyte in the liquid sample. The signal(s) at thereagent dots can be generated by any suitable reactions, such aschemical, biochemical, electrochemical, and/or binding reactionsinvolving the analyte, the reagents located at the reagent dots,reagents added to the liquid sample and/or other reagents dried on thetest device before use and are transported by the liquid sample or otherliquids to the reagent dots.

In another exemplary embodiment, the signal(s) at the reagent dots canbe generated by binding reactions involving the analyte and the reagentslocated at the reagent dots, and a labeled reagent added to the liquidsample or dried on the test device before use and is transported by theliquid sample or other liquids to the reagent dots. For example, themethod comprises a) contacting a liquid sample with the above testdevice, wherein the liquid sample is applied to a site of the testdevice upstream of the at least two of the reagent dots; b) transportingan analyte, if present in the liquid sample, and a labeled reagent tothe at least two of the reagent dots; and c) assessing the presence,absence, amount and/or pattern of signal(s) generated at the at leasttwo of the reagent dots to determining the presence, absence and/oramount of the analyte in the liquid sample.

In still another aspect, the present disclosure provides for a processfor manufacturing a test device for detecting an analyte in a liquidsample, which process comprises forming a plurality of reagent dots on amatrix to make a test device comprising at least two of said reagentdots that do not overlap and are sufficiently spaced apart from eachother so that when said liquid sample flows laterally along said matrix,flow of said liquid sample to, through and/or around one of said tworeagent dots does not substantially affect flow of said liquid sampleto, through and/or around said other reagent dot, wherein each of saidtwo reagent dots is neither a reagent line across the entire width ofsaid matrix in a direction perpendicular to the direction of said liquidsample flow nor a complete circle of a reagent line, and after a liquidsample flows laterally along said test device and passes said at leasttwo reagent dots, said at least two reagent dots form a predeterminedpattern to indicate presence, absence and/or amount of said analyte insaid liquid sample.

The principles of the present test devices and methods can be applied,or can be adapted to apply, to the lateral flow test devices and assaysknown in the art. For example, the principles of the present testdevices and methods can be applied, or can be adapted to apply, to thelateral flow test devices and assays disclosed and/or claimed in theU.S. Pat. Nos. 3,641,235, 3,959,078, 3,966,897, 4,094,647, 4,168,146,4,299,916, 4,347,312, 4,366,241, 4,391,904, 4,425,438, 4,517,288,4,960,691, 5,141,875, 4,857,453, 5,073,484, 4,695,554, 4,703,017,4,743,560, 5,075,078, 5,591,645, 5,656,448, RE 38,430 E, 5,602,040,6,017,767, 6,319,676, 6,352,862, 6,485,982, 5,120,643, 4,956,302, RE39,664 E, 5,252,496, 5,514,602, 7,238,538 B2, 7,175,992 B2, 6,770,487B2, 5,712,170, 5,275,785, 5,504,013, 6,156,271, 6,187,269, 6,399,398,7,317,532, EP 0,149,168 A1, EP 0,323,605 A1, EP 0,250,137 A2, GB1,526,708 and WO99/40438.

V. BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates schematic of a lateral flow test format.

FIG. 2( a) illustrates schematic of test development in sandwich format.FIG. 2( b) illustrates schematic of test development in competitiveformat.

FIG. 3( a) illustrates schematic of dot development with no affinitycapture reagent. FIG. 3( b) illustrates schematic of dot developmentwith low affinity capture reagent. FIG. 3( c) illustrates schematic ofdot development with capture reagent with high affinity for target.

FIG. 4 illustrates schematic of “Plus” sign development with the captureline in the direction of flow with a standard dispensing method.Development of a plus configuration with the test (capture) lineparallel to the direction of flow when a single continuous line ofreagent is used. Development of the line is incomplete due toperturbation of flow through the binding reagent area.

FIG. 5 illustrates schematic of the one dimensional nature of flow in alateral flow system and the effect of perturbation of flow by placementof a resistance in the flow path. Positioning a binding reagent featurein front of a line of binding reagent demonstrates the linear nature offlow in a lateral flow system. Binding to the blocking feature perturbsfluid flow, forcing fluid around the blockage and preventing thegeneration of a binding signal in the line of binding reagent behind theblockage. After a certain distance diffusion perpendicular to thedirection of flow occurs allowing for some development and finallycomplete development once the feature is positioned far enough away. Thedistance required for sufficient lateral diffusion to occur is dependenton the size of the blocking feature and the pore size of the porousmaterial.

FIG. 6 illustrates schematic of quantification array, 1 dimensional. Byplacing features of an appropriate size and distance from one anotherthe lateral flow of reagents is not perturbed between features so allfeatures can be exposed evenly to the migrating analyte. This allows forthe gradual binding of analyte and depletion of analyte concentration asit moves through the binding region. Changes in binding signal strengthfurther along the flow path are therefore indicative of analyteconcentration. This feature can be used to create quantitativethermometer-like results.

FIG. 7 illustrates schematic of 2-dimensional quantification array.Binding reagent is deposited in the flow path as pixels in twodimensions. The concentration of the binding reagent is the same in allpixels in the direction of flow, but the concentration changes in eachrow perpendicular to the direction of flow. The system acts to quantifyin the same manner as in FIG. 6, however, the titration of the bindingreagent in the second dimension allows for the generation of a greaterdynamic range in the assay

FIG. 8 illustrates schematic of multiplex array (4 analyte). Differentbinding reagents in each “channel” allows for multiplexing of analytedetection.

FIG. 9 illustrates an exemplary alpha numeric pixel array. Array can beprinted in any orientation, allowing for the creation of alpha-numericresults.

FIGS. 10A and 10B illustrate programmed and actual images of anexemplary signal readout. FIG. 10A illustrates programmed “X” symbol.FIG. 10B illustrates “X” symbol obtained from an actual test.

FIGS. 11A and 11B illustrate programmed and actual images of anexemplary signal readout. FIG. 11A illustrates programmed “+” symbol.FIG. 11B illustrates “+” symbol obtained from an actual test.

FIGS. 12A and 11B illustrate programmed and actual images of anotherexemplary signal readout. FIG. 12A illustrates programmed “+” symbol.FIG. 12B illustrates “+” symbol obtained from an actual test.

FIGS. 13A and 13B illustrate programmed and actual images of anexemplary signal readout. FIG. 13A illustrates programmed “X” symbol.FIG. 13B illustrates “X” symbol obtained from an actual test.

FIGS. 14A and 14B illustrate programmed and actual images of anexemplary signal readout. FIG. 14A illustrates programmed “TC” symbol.FIG. 14B illustrates “TC” symbol obtained from an actual test.

FIGS. 15A and 15B illustrate programmed and actual images of anexemplary signal readout. FIG. 15A illustrates another programmed “X”(within a box) symbol. FIG. 15B illustrates “X” (within a box) symbolobtained from an actual test.

FIGS. 16A and 16B illustrate programmed and actual images of anexemplary signal readout. FIG. 16A illustrates another programmed “YES”and “NO” symbol. FIG. 15B illustrates “YES” and “NO” symbol obtainedfrom an actual test.

FIG. 17 illustrates various symbols obtained from actual tests forimproving the clarity of signals.

FIG. 18 illustrates “X” symbol obtained from actual tests using afluorescent label (Europium).

FIGS. 19A-19E illustrate optimization of signal development using the“pixel” concept for lateral flow assays.

FIGS. 20A-20D illustrate applications of the “pixel” concept inmultiplexed lateral flow assays.

FIGS. 21A-21D illustrate construction of various spot patterns using asmaller dispense volume.

VI. DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, patentapplications (published or unpublished), and other publications referredto herein are incorporated by reference in their entireties. If adefinition set forth in this section is contrary to or otherwiseinconsistent with a definition set forth in the patents, applications,published applications and other publications that are hereinincorporated by reference, the definition set forth in this sectionprevails over the definition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, “the line is substantially parallel to the liquid sampleflow direction” means that the angle between the line and the liquidsample flow direction is at least less than 45 degrees or more than 135degrees. In some specific embodiments, the angle between the line andthe liquid sample flow direction is at about 40, 35, 30, 25, 20, 15, 10,5, 4, 3, 2, or 1 degree, or the line is completely parallel to theliquid sample flow direction. In other specific embodiments, the anglebetween the line and the liquid sample flow direction is at about 140,145, 150, 155, 160, 165, 170, 175, 176, 177, 178, or 179 degrees, or theline is completely parallel to the liquid sample flow direction.

As used herein, “the line is substantially perpendicular to the liquidsample flow direction” means that the angle between the line and theliquid sample flow direction is at least more than 45 degrees or lessthan 135 degrees. In some specific embodiments, the angle between theline and the liquid sample flow direction is at about 50, 55, 60, 65,70, 75, 80, 85, 86, 87 88 or 89 degrees, or the line is completelyperpendicular to the liquid sample flow direction. In other specificembodiments, the angle between the line and the liquid sample flowdirection is at about 130, 125, 120, 115, 110, 105, 100, 95, 94, 93, 92or 91 degrees, or the line is completely perpendicular to the liquidsample flow direction.

As used herein, “reagent dots have substantially the same size ordiameter” means that the difference in the size or diameter between thelargest dot and smallest dot is not more than one fold or less than 50%of the average or median size or diameter of the reagent dots. In somespecific embodiments, the difference in the size or diameter between thelargest dot and smallest dot is within 45%, 40%, 30%, 20%, 10%, 5%, or1% of the average or median size or diameter of the reagent dots. Inother specific embodiments, reagent dots have the same size or diameter.

As used herein, “the distance between reagent dots is substantially thesame” means that the distance between or among reagent dots, oftenadjacent reagent dots, is within 50% variation of the average or mediandistance between or among reagent dots or adjacent reagent dots. In somespecific embodiments, the distance between or among reagent dots oradjacent reagent dots is within 45%, 40%, 30%, 20%, 10%, 5%, or 1%variation of the average or median distance between or among reagentdots or adjacent reagent dots. In other specific embodiments, thedistance between or among reagent dots is the same. Such space ordistance can be measured by any suitable means. In some specificembodiments, the space or distance between or among reagent dots ismeasured as the space or distance between or among the edges of thereagent dots or adjacent reagent dots. In other specific embodiments,the space or distance between or among reagent dots is measured as thespace or distance between or among the centers or effective centers ofthe reagent dots or adjacent reagent dots.

As used herein, a “binding reagent” refers to any substance that bindsto target or analyte with desired affinity and/or specificity.Non-limiting examples of the binding reagent include cells, cellularorganelles, viruses, particles, microparticles, molecules, or anaggregate or complex thereof, or an aggregate or complex of molecules.Exemplary binding reagents can be an amino acid, a peptide, a protein,e.g., an antibody or receptor, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, e.g., DNA or RNA, a vitamin, amonosaccharide, an oligosaccharide, a carbohydrate, a lipid, an aptamerand a complex thereof.

As used herein, “antibody” includes not only intact polyclonal ormonoclonal antibodies, but also fragments thereof (such as Fab, Fab′,F(ab′)₂, Fv), single chain (ScFv), a diabody, a multi-specific antibodyformed from antibody fragments, mutants thereof, fusion proteinscomprising an antibody portion, and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity. An antibody includes an antibody of anyclass, such as IgG, IgA, or IgM (or sub-class thereof), and the antibodyneed not be of any particular class

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theantibodies comprising the population are identical except for possiblenaturally occurring mutations that are present in minor amounts. As usedherein, a “monoclonal antibody” further refers to functional fragmentsof monoclonal antibodies.

As used herein, the term “specifically binds” refers to the specificityof a binding reagent, e.g., an antibody, such that it preferentiallybinds to a defined analyte or target. Recognition by a binding reagentor an antibody of a particular analyte or target in the presence ofother potential targets is one characteristic of such binding. In someembodiments, a binding reagent that specifically binds to an analyteavoids binding to other interfering moiety or moieties in the sample tobe tested.

As used herein the term “avoids binding” refers to the specificity ofparticular binding reagents, e.g., antibodies or antibody fragments.Binding reagents, antibodies or antibody fragments that avoid binding toa particular moiety generally contain a specificity such that a largepercentage of the particular moiety would not be bound by such bindingreagents, antibodies or antibody fragments. This percentage generallylies within the acceptable cross reactivity percentage with interferingmoieties of assays utilizing the binding reagents or antibodies directedto detecting a specific target. Frequently, the binding reagents,antibodies or antibody fragments of the present disclosure avoid bindinggreater than about 90% of an interfering moiety, although higherpercentages are clearly contemplated and preferred. For example, bindingreagents, antibodies or antibody fragments of the present disclosureavoid binding about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, and about 99% or more of aninterfering moiety. Less occasionally, binding reagents, antibodies orantibody fragments of the present disclosure avoid binding greater thanabout 70%, or greater than about 75%, or greater than about 80%, orgreater than about 85% of an interfering moiety.

As used herein, “mammal” refers to any of the mammalian class ofspecies. Frequently, the term “mammal,” as used herein, refers tohumans, human subjects or human patients.

As used herein, the term “subject” is not limited to a specific speciesor sample type. For example, the term “subject” may refer to a patient,and frequently a human patient. However, this term is not limited tohumans and thus encompasses a variety of mammalian species.

As used herein the term “sample” refers to anything which may contain ananalyte for which an analyte assay is desired. The sample may be abiological sample, such as a biological fluid or a biological tissue.Examples of biological fluids include urine, blood, plasma, serum,saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus,amniotic fluid or the like. Biological tissues are aggregate of cells,usually of a particular kind together with their intercellular substancethat form one of the structural materials of a human, animal, plant,bacterial, fungal or viral structure, including connective, epithelium,muscle and nerve tissues. Examples of biological tissues also includeorgans, tumors, lymph nodes, arteries and individual cell(s).

As used herein, “stringency” of nucleic acid hybridization reactions isreadily determinable by one of ordinary skill in the art, and generallyis an empirical calculation dependent upon probe length, washingtemperature, and salt concentration. In general, longer probes requirehigher temperatures for proper annealing, while shorter probes needlower temperatures. Hybridization generally depends on the ability ofdenatured nucleic acid sequences to reanneal when complementary strandsare present in an environment below their melting temperature. Thehigher the degree of desired homology between the probe and hybridizablesequence, the higher the relative temperature that can be used. As aresult, it follows that higher relative temperatures would tend to makethe reaction conditions more stringent, while lower temperatures lessso. For additional details and explanation of stringency ofhybridization reactions, see Current Protocols in Molecular Biology(Ausubel et al. eds., Wiley Interscience Publishers, 1995); MolecularCloning: A Laboratory Manual (J. Sambrook, E. Fritsch, T. Maniatis eds.,Cold Spring Harbor Laboratory Press, 2d ed. 1989); Wood et al., Proc.Natl. Acad. Sci. USA, 82:1585-1588 (1985).

As used herein the term “isolated” refers to material removed from itsoriginal environment, and is altered from its natural state. Forexample, an isolated polypeptide could be coupled to a carrier, andstill be “isolated” because that polypeptide is not in its originalenvironment.

As used herein, “test substance (or candidate compound)” refers to achemically defined compound (e.g., organic molecules, inorganicmolecules, organic/inorganic molecules, proteins, peptides, nucleicacids, oligonucleotides, lipids, polysaccharides, saccharides, orhybrids among these molecules such as glycoproteins, etc.) or mixturesof compounds (e.g., a library of test compounds, natural extracts orculture supernatants, etc.) whose effect on a target is determined bythe disclosed and/or claimed methods herein.

As used herein, high-throughput screening (HTS) refers to processes thattest a large number of samples, such as samples of diverse chemicalstructures against disease targets to identify “hits” (see, e.g.,Broach, et al., High throughput screening for drug discovery, Nature,384:14-16 (1996); Janzen, et al., High throughput screening as adiscovery tool in the pharmaceutical industry, Lab Robotics Automation:8261-265 (1996); Fernandes, P. B., Letter from the society president, J.Biomol. Screening, 2:1 (1997); Burbaum, et al., New technologies forhigh-throughput screening, Curr. Opin. Chem. Biol., 1:72-78 (1997)). HTSoperations are highly automated and computerized to handle samplepreparation, assay procedures and the subsequent processing of largevolumes of data.

As used herein, “plant” refers to any of various photosynthetic,eucaryotic multi-cellular organisms of the kingdom Plantae,characteristically producing embryos, containing chloroplasts, havingcellulose cell walls and lacking locomotion.

As used herein, “animal” refers to a multi-cellular organism of thekingdom of Animalia, characterized by a capacity for locomotion,nonphotosynthetic metabolism, pronounced response to stimuli, restrictedgrowth and fixed bodily structure. Non-limiting examples of animalsinclude birds such as chickens, vertebrates such fish and mammals suchas mice, rats, rabbits, cats, dogs, pigs, cows, ox, sheep, goats,horses, monkeys and other non-human primates.

As used herein, “bacteria” refers to small prokaryotic organisms (lineardimensions of around 1 micron) with non-compartmentalized circular DNAand ribosomes of about 70S. Bacteria protein synthesis differs from thatof eukaryotes. Many anti-bacterial antibiotics interfere with bacteriaproteins synthesis but do not affect the infected host.

As used herein, “eubacteria” refers to a major subdivision of thebacteria except the archaebacteria. Most Gram-positive bacteria,cyanobacteria, mycoplasmas, enterobacteria, pseudomonas and chloroplastsare eubacteria. The cytoplasmic membrane of eubacteria containsester-linked lipids; there is peptidoglycan in the cell wall (ifpresent); and no introns have been discovered in eubacteria.

As used herein, “archaebacteria” refers to a major subdivision of thebacteria except the eubacteria. There are three main orders ofarchaebacteria: extreme halophiles, methanogens and sulphur-dependentextreme thermophiles. Archaebacteria differs from eubacteria inribosomal structure, the possession (in some case) of introns, and otherfeatures including membrane composition.

As used herein, “virus” refers to an obligate intracellular parasite ofliving but non-cellular nature, consisting of DNA or RNA and a proteincoat. Viruses range in diameter from about 20 to about 300 nm. Class Iviruses (Baltimore classification) have a double-stranded DNA as theirgenome; Class II viruses have a single-stranded DNA as their genome;Class III viruses have a double-stranded RNA as their genome; Class IVviruses have a positive single-stranded RNA as their genome, the genomeitself acting as mRNA; Class V viruses have a negative single-strandedRNA as their genome used as a template for mRNA synthesis; and Class VIviruses have a positive single-stranded RNA genome but with a DNAintermediate not only in replication but also in mRNA synthesis. Themajority of viruses are recognized by the diseases they cause in plants,animals and prokaryotes. Viruses of prokaryotes are known asbacteriophages.

As used herein, “fungus” refers to a division of eucaryotic organismsthat grow in irregular masses, without roots, stems, or leaves, and aredevoid of chlorophyll or other pigments capable of photosynthesis. Eachorganism (thallus) is unicellular to filamentous, and possesses branchedsomatic structures (hyphae) surrounded by cell walls containing glucanor chitin or both, and containing true nuclei.

B. Lateral Flow Devices Using Two Dimensional Features

In one aspect, the present disclosure provides for a test device fordetecting an analyte in a liquid sample, which device comprises aplurality of reagent dots on a matrix, wherein at least two of saidreagent dots do not overlap and are sufficiently spaced apart from eachother so that when said liquid sample flows laterally along said matrix,flow of said liquid sample to, through and/or around one of said tworeagent dots does not substantially affect flow of said liquid sampleto, through and/or around said other reagent dot, each of said tworeagent dots is neither a reagent line across the entire width of saidmatrix in a direction perpendicular to the direction of said liquidsample flow nor a complete circle of a reagent line, and after a liquidsample flows laterally along said test device and passes said at leasttwo reagent dots, said at least two reagent dots form a predeterminedpattern to indicate presence, absence and/or amount of said analyte insaid liquid sample.

Numerous variables can be considered to make the test device to ensurethat the reagent dots do not overlap and are sufficiently spaced apartfrom each other so that liquid sample flow to, through and/or around onereagent dot or set of reagent dots does not substantially affect flow ofthe liquid sample flow to, through and/or around other reagent dot orother sets of reagent dots. And at the same time, the test device shouldcomprise sufficient number of the reagent dots that can be used ingenerating signal readout to indicate presence, absence and/or amount ofsaid analyte in said liquid sample. Exemplary variables that can beconsidered and/or adjusted in making the test device include the numberof reagent dots, the size and/or shape of the reagent dots, e.g.,whether the absolute size or the size relative to the size of thematrix, the types and amounts of the reagents located at the reagentdots, the spacing between or among a portion or all reagent dots on thetest device, e.g., whether the absolute size of the spacing or the sizeof the spacing relative to the size of the matrix and/or the number ofthe reagent dots on the matrix, the orientation or position of thereagent dots relative to the liquid sample flow direction, theuniformity or variations of the sizes and/or shape among the reagentdots and the properties of the matrix, e.g., the material and/orporosity of the matrix, and/or the properties or composition of thesolution in which the reagent is spotted. Some or all of these variablescan be tested, adjusted or determined to make a test device that meetsthe intended test performance, e.g., meeting the intended or desiredassay sensitivity and/or specificity.

In some specific embodiments, it can be determined that the reagent dotsdo overlap and are not sufficiently spaced apart from each other so thatliquid sample flow to, through and/or around one reagent dot or set ofreagent dots blocks or prevents flow of the liquid sample flow to,through and/or around other reagent dot or other sets of reagent dots.Some or all of these variables can then be adjusted so that the liquidsample flow blocking effect be reduced by at least 10%, and preferablyby at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,99%, or 100%. In other specific embodiments, given a particularconfiguration, the liquid sample flow to, through and/or around otherreagent dot or other sets of reagent dots can be determined. Some or allof these variables can then be adjusted so that the liquid sample flowto, through and/or around other reagent dot or other sets of reagentdots be increased by at least 10%, and preferably by at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In otherembodiments, some or all of these variables can then be adjusted so thatthe liquid sample flow to, through and/or around other reagent dot orother sets of reagent dots be increased by 1 fold, 2 folds, 3 folds, 4folds, 5 folds 6 folds, 7 folds, 8 folds, 9 folds, 10 folds, or more.

The test device can comprise any suitable number of reagent dots. In oneexample, the test device comprises two reagent dots. In another example,the test device comprises more than two reagent dots, such as at least3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1,000, 5,000, 10,000 or morereagent dots.

Any suitable number, portion or all of the reagent dots in the testdevice can be sufficiently spaced apart from each other. For example, atleast a quarter, a third, half or all reagent dots do not overlap andare sufficiently spaced apart from each other so that when the liquidsample flows laterally along the matrix, flow of the liquid sample to,through and/or around one of the reagent dots does not substantiallyaffect flow of the liquid sample to, through and/or around the otherreagent dots.

The predetermined pattern formed at the reagent dots can take any form,shape and/or pattern. For example, the predetermined pattern can be aline, multiple lines, a symbol, a geometric shape and an alpha-numericshape, a regular shape, or a irregular shape, or a combination thereof.The exemplary regular shape can be a line, a circle, a rod, a square, atriangle, and a rectangle. The exemplary alpha-numeric shape can be aletter, a word, a number or a combination thereof.

When the predetermined pattern is in the form of a line or multiplelines, the line(s) can be at any suitable orientation or positionrelative to the liquid sample flow direction. In one example, theline(s) is substantially parallel to the liquid sample flow direction.In another example, the line(s) is substantially perpendicular to theliquid sample flow direction. In still another example, thepredetermined pattern is in the form of multiple lines. The multiplelines can comprise at least a line that is substantially parallel to theliquid sample flow direction and at least a line that is substantiallyperpendicular to the liquid sample flow direction. In some embodiments,at least a quarter, a third, half of the lines are substantiallyparallel to the liquid sample flow direction. In other embodiments, atleast a quarter, a third, half of the lines are substantiallyperpendicular to the liquid sample flow direction.

The test device can be used to detect a single analyte or multipleanalytes in a liquid sample. In one example, the plurality of reagentdots in the test device comprises different reagents and the test deviceis used to detect multiple analytes in the liquid sample. In anotherexample, the plurality of reagent dots in the test device comprises thesame reagent and the test device is used to detect the amount of asingle analyte in the liquid sample.

The reagent dots in the test device can comprise any suitable amount ofthe reagent(s). In one example, the plurality of reagent dot comprisesthe same amount of the reagent(s). In another example, the plurality ofreagent dots comprises the different amounts of the reagent(s).

The reagent dots in the test device can have any suitable size(s). Inone example, at least one of the reagent dots has a diameter of about0.1-1 um, 1-10 um, 10-50 um, 51-100 um, 101-200 um, 201-300 um, 301-400um, 401-500 um and 501-1000 um. In another example, at least a quarter,a third, half or all reagent dots have a diameter of about 0.1-1 um,1-10 um, 10-50 um, 51-100 um, 101-200 um, 201-300 um, 301-400 um,401-500 um or 501-1000 um. In still another example, at least one of thereagent dots has a diameter or surface area that is about 10%, 5%, 1%,0.5%, 0.1%, 0.05%, 0.01%, 0.001% or smaller diameter or surface area ofthe length, width or surface area of the matrix calculated by the widthand length of the membrane. In yet another example, at least a quarter,a third, half or all reagent dots have a diameter or surface area thatis about 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or smallerdiameter or surface area of the length, width or surface of the matrix.

Any suitable drop volumes can be used to make spots with any suitable ordesirable sizes. In exemplary embodiments, the range of drop volumesused to create the range of spot sizes on the flow membrane can be inthe range of about 30-200 pL, 201-500 pL, 501 pL-1.001 mL, 1.001 mL to5.0 mL, 5.1-25 mL, 21.1-100 mL, or 100.1-500 mL. Shown in the belowTable 1 is both spherical and hemispherical diameter of various dropsizes in the above drop range.

TABLE 1 Drop Sphere Hemisphere Volume Diameter(um) Size (um) 1 pL 12.415.3 10 26.7 33.8 100 58 72 500 98 124 1 nL 124 156 2.08 158 199 5 212268 10 266 337 20 336 423 50 457 577 100 575 725 500 982 1243The actual developed spot size of a reagent drop on the membrane can belarger, e.g., about 10-25% larger, than the hemispherical drop diameter.The sphere and hemispherical size of different drop volumes with therange described above is shown in the above Table 1.

The meaning of a “diameter” is often determined by the shape of the dot.For example, if the dot is a circle, the diameter of a circle is anystraight line segment that passes through the center of the circle andwhose endpoints are on the circle. The length of a diameter is alsocalled the diameter. For a convex shape in the plane, the diameter isdefined to be the largest distance that can be formed between twoopposite parallel lines tangent to its boundary. The use of “diameter”does not limit the dot shape to be a circle or other regular shape. Insome specific embodiments, when a dot has an irregular shape, a“diameter” can be measured as a parameter that indicates the length orwidth of the dot, e.g., measured as the largest distance between twopoints on the dot.

The reagent dots in the test device can have the same or differentsize(s) or diameter(s). In one example, at least a quarter, a third,half or all reagent dots have substantially the same size or diameter.In another example, at least a quarter, a third, half or all reagentdots have substantially different sizes or diameters.

The reagent dots in the test device can have any suitable shapes, e.g.,any suitable regular or irregular shape. In one example, at least one ofthe reagent dots has a shape that is a line, a circle, a rod, a square,a triangle, a rectangle or an irregular shape. In another example, atleast a quarter, a third, half or all reagent dots have a shape that isa line, a circle, a rod, a square, a triangle, a rectangle or anirregular shape. The reagent dots in the test device can have the sameor different shape(s). In one example, at least a quarter, a third, halfor all reagent dots have the same shape. In another example, at least aquarter, a third, half or all reagent dots have different shapes.

The reagent dots can have any suitable space(s) or distance(s) betweenor among the dots. In one example, the distance between or among thereagent dots is about 1-10 um, 10-50 um, 51-100 um, 101-200 um, 201-300um, 301-400, 401-500, or 501-600 um. The space(s) or distance(s) betweenor among the reagent dots can be the same or different. In one example,the space or distance between at least a quarter, a third, half or allreagent dots is substantially the same. In another example, the space ordistances between at least a quarter, a third, half or all reagent dotsare different. Such space or distance can be measured by any suitablemeans. In some specific embodiments, the space or distance between oramong reagent dots is measured as the space or distance between or amongthe edges of the reagent dots or adjacent reagent dots, e.g., distancebetween or among the edges of dots which defines the low resistance flowpath of reagents. In other specific embodiments, the space or distancebetween or among reagent dots is measured as the space or distancebetween or among the centers or effective centers of the reagent dots oradjacent reagent dots.

The reagent dots can be located on any suitable places or side(s) of thematrix. In one example, the test device comprises a single layer of theplurality of reagent dot. In another example, the test device comprisesmultiple layers, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more layers, of theplurality of reagent dots. In still another example, the test devicecomprises at least a layer of the plurality of reagent dots on one sideof the matrix. In yet another example, the test device comprises atleast a layer of the plurality of reagent dots on both sides of thematrix.

The signal(s) at the reagent dots can be generated by any suitablereactions, such as chemical, biochemical, electrochemical, and/orbinding reactions involving the analyte, the reagents located at thereagent dots, reagents added to the liquid sample and/or otherliquid(s), and/or other reagents dried on the test device before use andthat are transported by the liquid sample or other liquids to thereagent dots.

In some embodiments, the signal(s) at the reagent dots are generatedbased on binding reactions involving the analyte, the reagents locatedat the reagent dots, reagents added to the liquid sample and/or otherliquid(s), and/or other reagents dried on the test device before use andthat are transported by the liquid sample or other liquids to thereagent dots. In one example, at least one of the reagent dots comprisesa reagent that is capable of binding to an analyte or another bindingreagent that is capable of binding to an analyte. Preferably, thereagent is capable of specifically binding to an analyte or anotherbinding reagent that is capable of binding to an analyte. Alsopreferably, the reagent avoids binding to interfering moiety or moietiesin the testing sample. In another example, at least a quarter, a third,half or all reagent dots comprise a reagent that is capable of bindingto an analyte or another binding reagent that is capable of binding toan analyte. Preferably, the reagents are capable of specifically bindingto an analyte or another binding reagent that is capable of binding toan analyte.

The reagents located at the reagent dots can be any suitable substances.For example, the reagents can be inorganic molecules, organic moleculesor complexes thereof. Exemplary inorganic molecules can be ions such assodium, potassium, magnesium, calcium, chlorine, iron, copper, zinc,manganese, cobalt, iodine, molybdenum, vanadium, nickel, chromium,fluorine, silicon, tin, boron or arsenic ions. Exemplary organicmolecules can be an amino acid, a peptide, a protein, e.g., an antibodyor receptor, a nucleoside, a nucleotide, an oligonucleotide, a nucleicacid, e.g., DNA or RNA, a vitamin, a monosaccharide, an oligosaccharide,a carbohydrate, a lipid and a complex thereof.

Exemplary amino acids can be a D- or a L-amino-acid. Exemplary aminoacids can also be any building blocks of naturally occurring peptidesand proteins including Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln(Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe(F), Pro (P) Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V).

Any suitable proteins or peptides can be used as the reagents on thetest device. For example, enzymes, transport proteins such as ionchannels and pumps, nutrient or storage proteins, contractile or motileproteins such as actins and myosins, structural proteins, defenseprotein or regulatory proteins such as antibodies, hormones and growthfactors can be used. Proteineous or peptidic antigens can also be used.

Any suitable nucleic acids, including single-, double andtriple-stranded nucleic acids, can be used as the reagents on the testdevice. Examples of such nucleic acids include DNA, such as A-, B- orZ-form DNA, and RNA such as mRNA, tRNA and rRNA.

Any suitable nucleosides can be can be used as the reagents on the testdevice. Examples of such nucleosides include adenosine, guanosine,cytidine, thymidine and uridine. Any nucleotides can be used as thereagents on the test device. Examples of such nucleotides include AMP,GMP, CMP, UMP, ADP, GDP, CDP, UDP, ATP, GTP, CTP, UTP, dAMP, dGMP, dCMP,dTMP, dADP, dGDP, dCDP, dTDP, dATP, dGTP, dCTP and dTTP.

Any suitable vitamins can be used as the reagents on the test device.For example, water-soluble vitamins such as thiamine, riboflavin,nicotinic acid, pantothenic acid, pyridoxine, biotin, folate, vitaminB₁₂ and ascorbic acid can be used. Similarly, fat-soluble vitamins suchas vitamin A, vitamin D, vitamin E, and vitamin K can be used.

Any suitable monosaccharides, whether D- or L-monosaccharides andwhether aldoses or ketoses, can be used as the reagents on the testdevice. Examples of monosaccharides include triose such asglyceraldehyde, tetroses such as erythrose and threose, pentoses such asribose, arabinose, xylose, lyxose and ribulose, hexoses such as allose,altrose, glucose, mannose, gulose, idose, galactose, talose and fructoseand heptose such as sedoheptulose.

Any suitable lipids can be used as the reagents on the test device.Examples of lipids include triacylglycerols such as tristearin,tripalmitin and triolein, waxes, phosphoglycerides such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol and cardiolipin, sphingolipids such assphingomyelin, cerebrosides and gangliosides, sterols such ascholesterol and stigmasterol and sterol fatty acid esters. The fattyacids can be saturated fatty acids such as lauric acid, myristic acid,palmitic acid, stearic acid, arachidic acid and lignoceric acid, or canbe unsaturated fatty acids such as palmitoleic acid, oleic acid,linoleic acid, linolenic acid and arachidonic acid.

In one specific embodiment, the analyte to be detected comprises or isan antigen, the binding reagent on the test device comprises or is anantibody. Preferably, the antibody specifically binds to the analyte. Inone example, the test device is used in a sandwich assay format, inwhich a binding reagent, e.g., an antibody, is used as a reagent at thereagent dots, and another binding reagent having a detectable label isalso used to form a labeled binding reagent-analyte-binding reagent orantibody sandwich at the reagent dots to generate readout signals.Alternatively, a binding reagent is used as a reagent at the reagentdots, and an antibody have a detectable label is also used to form alabeled antibody-analyte-binding reagent sandwich at the reagent dots togenerate readout signals. In one example, the sandwich assay uses twoantibodies, one as the capture reagent and the other as the labeledreagent.

The test device can also used in a competition assay format. In oneexample, a binding reagent, e.g., an antibody, is used as a capturereagent at the reagent dots. An analyte or analyte analog having adetectable label, either added in a liquid or previously dried on thetest device and redissolved or resuspnded by a liquid, will compete withan analyte in a sample to bind to the capture reagent at the reagentdots. In another example, an analyte or analyte analog is used as acapture reagent at the reagent dots. A binding reagent, e.g., anantibody, having a detectable label, is either added in a liquid orpreviously dried on the test device and redissolved or resuspnded by aliquid. An analyte in a sample will compete with the analyte or analyteanalog at the reagent dots for binding to the binding reagent, e.g., anantibody, having a detectable label.

The matrix can have any suitable structure. In one example, the matrixcan have a porous structure. The matrix can comprise any suitablematerial(s). For example, porous plastics material, such aspolypropylene, polyethylene (preferably of very high molecular weight),polyvinylidene flouride, ethylene vinylacetate, acrylonitrile andpolytetrafluoro-ethylene can be used. See e.g., U.S. Pat. No. 6,187,598.It can be advantageous to pre-treat the member with a surface-activeagent during manufacture, as this can reduce any inherent hydrophobicityin the member and therefore enhance its ability to take up and deliver amoist sample rapidly and efficiently. The matrix can also be made frompaper or other cellulosic materials. In some embodiments, the matrixcomprises or is made of nitrocellulose or glass fiber.

In another example, the matrix can have a non-porous structure, e.g.,plastic solid surface. In some embodiments, the matrix can have otherstructures such as channels or other guided fluid pathways. In anotherexample, the matrix comprises a plastic, a film of a matrix having ahydrophilic surface, or a material with a controlled contact angle withthe sample liquid.

The reagent dots can comprise any suitable reagents and can be arrangedto form any suitable pattern. In one example, the plurality of reagentdots comprises the same binding reagent that is capable of binding to ananalyte or another binding reagent that is capable of binding to theanalyte. The plurality of reagent dots form a line that is substantiallyparallel to the liquid sample flow direction. As the liquid sample flowslaterally along the test device, the analyte, if present in the liquidsample, becomes sequentially bound to the binding reagent at each of thereagent dots until the analyte is depleted by binding to the upstreamreagent dot(s). The binding of the analyte to the reagent dot(s)generates a dateable signal at the reagent dot(s), and the intensityand/or the number of the dateable signal at the reagent dot(s) providesa quantitation or a semi-quantitation of the analyte in the liquidsample.

In another example, the plurality of reagent dots comprises differentbinding reagents that are capable of binding to different analytes orother binding reagents that are capable of binding to the analytes. Theplurality of reagent dots forms a line that is substantially parallel tothe liquid sample flow direction. As the liquid sample flows laterallyalong the test device, the analytes, if present in the liquid sample,become bound to the binding reagents at each of the reagent dots. Thebinding of the analytes to the reagent dots generates dateable signalsat the reagent dots, and the presence and/or intensity of the dateablesignals at the reagent dots indicates the presence and/or amount of theanalytes in the liquid sample.

In still another example, the plurality of reagent dots comprisesdifferent groups of binding reagents, each group of the binding reagentsis capable of binding to the same analyte or another binding reagentthat is capable of binding to the same analyte, and the binding reagentsin different groups are capable of binding to different analytes orother binding reagents that are capable of binding to differentanalytes. Each group of the reagent dots forms a line that issubstantially parallel to the liquid sample flow direction, and thedifferent lines formed by the different groups of the reagent dots aresubstantially parallel to each other. As the liquid sample flowslaterally alone the test device, the analytes, if present in the liquidsample, become sequentially bound to the binding reagents at each of thereagent dots in each group of the reagent dots until the analytes aredepleted by binding to the upstream reagent dots. The binding of theanalytes to the reagent dots generates dateable signals at the reagentdots, and the intensity and/or the number of the dateable signals at thereagent dots provides a quantitation or a semi-quantitation of thedifferent analytes in the liquid sample.

In yet another example, the plurality of reagent dots comprises the samebinding reagent that is capable of binding to an analyte or anotherbinding reagent that is capable of binding to the analyte. The pluralityof reagent dots forms multiple lines that are substantially parallel tothe liquid sample flow direction. The reagent dots in each line comprisethe same amount of the binding reagent, but the reagent dots indifferent lines comprise the different amounts of the binding reagent.As the liquid sample flows laterally alone the test device, the analyte,if present in the liquid sample, becomes sequentially bound to thebinding reagent at each of the reagent dots in each of the lines untilthe analyte is depleted by binding to the upstream reagent dot(s) ineach of the lines. The binding of the analyte to the reagent dot(s)generates a dateable signal at the reagent dot(s), and the intensityand/or the number of the dateable signal at the reagent dot(s) providesa quantitation or a semi-quantitation of the analyte in the liquidsample. The reagent dots in different lines can comprise the same ordifferent amounts of the binding reagent. In one embodiment, from oneend to the other end of the test device, in the direction perpendicularto the direction of said liquid sample flow, the reagent dots indifferent lines comprise the sequentially different amounts of thebinding reagent, e.g., sequentially increasing or decreasing amounts ofthe binding reagent.

In yet another example, the plurality of reagent dots comprises twodifferent groups of binding reagents. One group of the reagent dotsforms a line that is at a first angle relative to the liquid sample flowdirection, and the other group of the reagent dots forms a line that isat a second, different angle relative to the liquid sample flowdirection. After the liquid sample flows laterally along the testdevice, the reagent dots in one of the lines generate a signalindicating the presence and/or amount of an analyte in the liquidsample, and the reagent dots in the other line generate a control signalindicating the test is properly conducted. When the liquid samplecomprises the analyte and the test is properly conducted, the two linesof the reagent dots generate a positive symbol, indicating the presenceand/or amount of the analyte in the liquid sample. When the liquidsample does not comprise the analyte and the test is properly conducted,only one line of the reagent dots generates a negative symbol,indicating the absence of the analyte in the liquid sample.

The two different groups of binding reagents can form lines that are atany suitable angles relative to the liquid sample flow direction, andthe reagent dots can form any suitable readout signals to indicate thepresence, absence and/or amount of the analyte in the liquid sample. Forexample, one group of the reagent dots forms a line that issubstantially parallel to the liquid sample flow direction, and theother group of the reagent dots forms a line that is substantiallyperpendicular to the liquid sample flow direction. When the liquidsample comprises the analyte and the test is properly conducted, the twolines of the reagent dots generate a “+” symbol, indicating the presenceand/or amount of the analyte in the liquid sample, and when the liquidsample does not comprise the analyte and the test is properly conducted,only one line of the reagent dots generates a “−” symbol, indicating theabsence of the analyte in the liquid sample.

In yet another example, the plurality of reagent dots comprises twodifferent groups of binding reagents. After the liquid sample flowslaterally along the test device, reagent dots in one group generate analpha-numeric signal indicating the presence and/or amount of an analytein the liquid sample, and the reagent dots in the other group generate acontrol symbol signal indicating the test is properly conducted. Thealpha-numeric signal can take any suitable forms. For example, thealpha-numeric signal can be a word such as yes, Pos, Positive, Neg,Negative, No, or OK. The control symbol signal can also take anysuitable forms. For example, the control symbol signal can be a “+”sign. The test device can be configured for any suitable form of test,e.g., a sandwich or competitive test.

In yet another example, the plurality of reagent dots comprises areagent that binds to an intended binder and the binding pattern betweenthe reagent and the binder formed on the reagent dots indicates akinetic property of the binding between the reagent and the binder. Thereagent and the intended binder can be any suitable or desiredsubstances. For example, the reagent can be an antigen, the binder canbe an antibody to the antigen, or vice versa. The binding patternbetween the antigen and the antibody formed on the reagent dots canindicate a kinetic property of the binding between the antigen and theantibody. The present device can be used to indicate any suitablekinetic property of the binding between the antigen and the antibody.For example, the kinetic property can comprise the binding affinity ofthe binding between the antigen and the antibody. The binding affinitycan be detected with any suitable signal readout. For example, thebinding pattern of a leading edge on the dot(s) can indicate that thebinding reagent or antibody has a high binding affinity for the antigen,a filled out but generally inconsistent dot indicating a low affinitybinding reagent, and a blanking spot indicating no specific bindingbetween the binding reagent or antibody and the antigen. See e.g., FIG.3.

In yet another example, the test device can comprise at least one groupof the reagent dots that generate an additional signal that is notrelated to the presence, absence and/or amount of the analyte in theliquid sample, or whether the test is properly conducted. Suchadditional reagent dots can be used for any suitable purposes. Forexample, the additional signal can be used to indicate the authenticity,quality and/or identification of the test device, or identification ofthe liquid sample. The additional signal can have any suitable form orpattern. For example, the additional signal can comprise analpha-numeric signal.

In yet another example, the test device can comprise at least one groupof the reagent dots that form a circle around the sample applicationlocation, and the liquid sample moves radially to pass the group of thereagent dots. In yet another example, the test device can furthercomprise a flow through device portion.

The matrix can have any suitable form or shape. For example, the matrixcan be in the form of a strip or a circle. The matrix can also havesuitable number of elements. For example, the matrix can be made of asingle element or can comprise multiple elements.

The test device can further comprise a sample application elementupstream from and in fluid communication with the matrix. The sampleapplication element can be made of any suitable materials, such asnitrocellulose, glass fiber, polypropylene, polyethylene (preferably ofvery high molecular weight), polyvinylidene flouride, ethylenevinylacetate, acrylonitrile or polytetrafluoro-ethylene. The matrix andthe sample application element can comprise the same or differentmaterials.

The test device can further comprise a liquid absorption elementdownstream from and in fluid communication with the matrix. The liquidabsorption element can be made of any suitable materials, such as paperor cellulose materials.

The test device can further comprise a control location comprising meansfor indicating proper flow of the liquid sample and/or a valid testresult. Any suitable means can be used. In one example, the meanscomprises a binding reagent that binds to a binding reagent with adetectable label that also binds to the analyte. In another example, themeans comprises a binding reagent that binds to a binding reagent with adetectable label that does not bind to the analyte. In still anotherexample, the means comprises a substance that will generate a detectablesignal, e.g., color or electrical signal, once a liquid flow along orthrough the control location.

In some embodiments, at least a portion of the matrix is supported by asolid backing. In other embodiments, half, more than half or all portionof the matrix is supported by a solid backing. The solid backing can bemade of any suitable material, e.g., solid plastics. If the test devicecomprises electrode or other electrical elements, the solid backingshould generally comprise non-conductive materials.

In some embodiments, a labeled reagent can be dried on the test deviceand the dried labeled reagent can be redissolved or resuspended by aliquid, e.g., a sample liquid and/or additional liquid, and transportedlaterally through the test device to generate readout, control and/orother signals. For example, a portion of the matrix, upstream from theat least two of the reagent dots, can comprise a dried, labeled reagent,the labeled reagent capable of being moved by a liquid sample and/or afurther liquid to the at least two of the reagent dots and/or a controllocation to generate a detectable signal. The dried, labeled reagent canbe located at any suitable places on the test device. In one example,the dried, labeled reagent is located downstream from a sampleapplication place on the test device. In another example, the dried,labeled reagent is located upstream from a sample application place onthe test device. The type of the labeled reagent can be determined basedon the intended assay formats. For example, if the test device is to beused in a sandwich assay, the labeled reagent should be capable ofbinding, and preferably capable of specifically binding, to the analyteor another substance that binds to the analyte. The same labeled reagentcan also be used for certain competitive binding assays. For other typesof the competitive binding assays, the labeled reagent should be ananalyte or an analyte analog linked to a detectable label.

In some embodiments, the test device can further comprise, upstream fromthe at least two of the reagent dots, a conjugate element that comprisesa dried, labeled reagent, the labeled reagent being capable of moved bya liquid sample and/or a further liquid to the at least two of thereagent dots and/or a control location to generate a detectable signal.The conjugate element can be located downstream from a sampleapplication place on the test device. The conjugate element can also belocated upstream from a sample application place on the test device. Insome embodiments, the labeled reagent binds to an analyte in the liquidsample. In other embodiments, the labeled reagent competes with ananalyte in the liquid sample for binding to a binding reagent for theanalyte at the at least two of the reagent dots.

Any suitable label can be used. The label can be a soluble label, suchas a colorimetric, radioactive, enzymatic, luminescent or fluorescentlabel. The label can also be a particle or particulate label, such as aparticulate direct label, or a colored particle label. Exemplaryparticle or particulate labels include colloidal gold label, latexparticle label, nanoparticle label and quantum dot label. Depending onthe specific configurations, the labels such as colorimetric,radioactive, enzymatic, luminescent or fluorescent label, can be eithera soluble label or a particle or particulate label.

In some embodiments, the labeled reagent is dried in the presence of amaterial that stabilizes the labeled reagent, facilitates solubilizationor resuspension of the labeled reagent in a liquid, and/or facilitatesmobility of the labeled reagent. Any suitable material can be used. Forexample, the material can be a protein, e.g., a meta-soluble protein, apeptide, a polysaccharide, a sugar, e.g., sucrose, a polymer, a gelatinor a detergent. See e.g., U.S. Pat. Nos. 5,120,643 and 6,187,598.

The present test devices can be used with any suitable sample liquid. Inone example, a sample liquid alone is used to transport the analyteand/or the labeled reagent to the at least two of the reagent dots. Inanother example, a developing liquid is used to transport the analyteand/or the labeled reagent to the at least two of the reagent dots. Instill another example, both sample liquid and a developing liquid isused to transport the analyte and/or the labeled reagent to the at leasttwo of the reagent dots.

In some embodiments, the test device can further comprise a housing thatcovers at least a portion of the test device, wherein the housingcomprises a sample application port to allow sample application upstreamfrom or to the at least two of the reagent dots and an optic openingaround the at least two of the reagent dots to allow signal detection atthe two of the reagent dots. The optic opening can be achieved in anysuitable way. For example, the optic opening can simply be an openspace. Alternatively, the optic opening can be a transparent cover.

In other embodiments, the housing can cover the entire test device. Instill other embodiments, at least a portion of the sample receivingportion of the matrix or the sample application element is not coveredby the housing and a sample is applied to the portion of the samplereceiving portion of the matrix or the sample application elementoutside the housing and then transported to the at least two of thereagent dots. The housing can comprise any suitable material. Forexample, the housing can comprise a plastic material, a biodegradablematerial or a cellulosic material. In another example, the housing,whether in part or in its entirety, can comprise an opaque, translucentand/or transparent material.

In some embodiments, the present invention provides for a test devicewherein the liquid sample has moved laterally along the test device togenerate detectable signal(s) at the at least two of the reagent dots.

C. Methods for Detecting an Analyte Using a Lateral Flow Device with TwoDimensional Features

In another aspect, the present disclosure provides for a method fordetecting an analyte using the above test device. In one exemplaryembodiment, the present disclosure provides for a method for detectingan analyte in a liquid sample, which method comprises a) contacting aliquid sample with the above test device, wherein the liquid sample isapplied to a site of the test device upstream of the at least two of thereagent dots; b) transporting an analyte, if present in the liquidsample, to the at least two of the reagent dots; and c) assessing thepresence, absence, amount and/or pattern of signal(s) generated at theat least two of the reagent dots to determining the presence, absenceand/or amount of the analyte in the liquid sample. The signal(s) at thereagent dots can be generated by any suitable reactions, such aschemical, biochemical, electrochemical, and/or binding reactionsinvolving the analyte, the reagents located at the reagent dots,reagents added to the liquid sample and/or other reagents dried on thetest device before use and are transported by the liquid sample or otherliquids to the reagent dots.

In another exemplary embodiment, the signal(s) at the reagent dots canbe generated by binding reactions involving the analyte and the reagentslocated at the reagent dots, and a labeled reagent added to the liquidsample or dried on the test device before use and is transported by theliquid sample or other liquids to the reagent dots. For example, themethod comprises a) contacting a liquid sample with the above testdevice, wherein the liquid sample is applied to a site of the testdevice upstream of the at least two of the reagent dots; b) transportingan analyte, if present in the liquid sample, and a labeled reagent tothe at least two of the reagent dots; and c) assessing the presence,absence, amount and/or pattern of signal(s) at the at least two of thereagent dots, e.g., signal(s) generated by the labeled reagent at the atleast two of the reagent dots, to determining the presence, absenceand/or amount of the analyte in the liquid sample.

In some embodiments, the liquid sample and the labeled reagent arepremixed to form a mixture and the mixture is applied to the testdevice. For example, the labeled reagent can be provided or stored in aliquid and then can be premixed with a sample liquid to form a mixtureand the mixture is applied to the test device. In another example, thelabeled reagent can be dried in a location or container not in fluidcommunication with the test device, e.g., in a test tube or well such asa microtiter plate well. In use, the sample liquid can be added to thecontainer, e.g., the test tube or well, to form the mixture and themixture can then be applied to the test device.

In other embodiments, the test device comprises a dried labeled reagentbefore use and the dried labeled reagent is solubilized or resuspended,and transported to the at least two of the reagent dots by the liquidsample and/or other liquid. The dried labeled reagent can be located atany suitable location on the test device. For example, the dried labeledreagent can be located downstream from the sample application site, andthe dried labeled reagent can be solubilized or resuspended, andtransported to the at least two of the reagent dots by the liquid sampleand/or other liquid. In another example, the dried labeled reagent canbe located upstream from the sample application site, and the driedlabeled reagent can be solubilized or resuspended, and transported tothe at least two of the reagent dots by another liquid.

In some embodiments, the labeled reagent is solubilized or resuspended,and transported to the at least two of the reagent dots by the liquidsample alone. In other embodiments, the analyte and/or labeled reagentis solubilized or resuspended, and transported to the at least two ofthe reagent dots by another liquid. In still other embodiments, theanalyte and/or labeled reagent is solubilized or resuspended, andtransported to the at least two of the reagent dots by both the sampleliquid and another liquid, e.g., a developing liquid.

The present test devices can be used to detect an analyte in anysuitable sample liquid. In some embodiments, the liquid sample can bebody fluid sample, such as a whole blood, a serum, a plasma, a urinesample or an oral fluid. Such body fluid sample can be sued directly orcan be processed, e.g., enriched, purified, or diluted, before use. Inother embodiments, the liquid sample can be a liquid extract, suspensionor solution derived from a solid or semi-solid biological material suchas a phage, a virus, a bacterial cell, an eukaryotic cell, a fugal cell,a mammalian cell, a cultured cell, a cellular or subcellular structure,cell aggregates, tissue or organs. In specific embodiments, the sampleliquid is obtained or derived from a mammalian or human source. In stillother embodiments, the liquid sample is a sample derived from abiological, a forensics, a food, a biowarfare, or an environmentalsource. In other embodiments, the sample liquid is a clinical sample,e.g., a human or animal clinical sample. In still other embodiments, thesample liquid is a man-made sample, e.g., a standard sample for qualitycontrol or calibration purposes.

The present test devices can be used to detect the presence, absenceand/or amount of an analyte in any suitable sample liquid. In someembodiments, the present test devices are used to detect the presence orabsence of an analyte in any suitable sample liquid, i.e., to provide ayes or no answer. In other embodiments, the present test devices areused to quantify or semi-quantify the amount of an analyte in a liquidsample.

The present test devices can be used to detect the presence, absenceand/or amount of a single analyte in any suitable sample liquid.Alternatively, the present test devices can be used to detect thepresence, absence and/or amount of multiple analytes in a liquid sample.In still other embodiments, the present test devices can be used toquantify or semi-quantify the amounts of the multiple analytes in theliquid sample.

The present test devices can be used to detect the presence, absenceand/or amount of any suitable analyte in a sample liquid. Exemplaryanalytes include inorganic molecules, organic molecules or complexesthereof. Exemplary inorganic molecules can be ions such as sodium,potassium, magnesium, calcium, chlorine, iron, copper, zinc, manganese,cobalt, iodine, molybdenum, vanadium, nickel, chromium, fluorine,silicon, tin, boron or arsenic ions. Exemplary organic molecules can bean amino acid, a peptide, a protein, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, e.g., a DNA or RNA molecule or a hybridthereof, a vitamin, a monosaccharide, an oligosaccharide, acarbohydrate, a lipid and a complex thereof. In some embodiments, theanalyte is a cell, a virus or a molecule. In other embodiments, theanalyte is hCG, hLH, hFSH, hTSH, a disease or disorder marker, e.g., acardiac biomarker, an antigen of an infectious organism, an antibody toan infectious organism, etc.

The present methods can be used for any suitable purpose. For example,present methods can be used for clinical diagnosis, prognosis, riskassessment and prediction, stratification and treatment monitoring andadjustment. In another example, present methods can be used for variousresearch purposes, such as basic research, drug candidate screening,animal studies, and clinical trials. In still another example, presentmethods can be used in tests for standard setting, quality control,illegal drug screening, food safety, environmental safety, industrialsafety, pollution, detection of biowarfare agents, screening for drugsor pharmaceuticals, and monitoring the quality of manufacturing usingbioreactors looking for unwanted molecules, etc. The present testsdevices and methods can be used in any suitable settings, such as testsin the labs, clinics, hospitals, physician's offices, homes, naturalenvironments, battle fields and first responder environments, e.g.,environments for fire, paramedic, police actions.

D. Process for Manufacturing a Lateral Flow Device With Two DimensionalFeatures

In still another aspect, the present disclosure provides for a processfor manufacturing a test device for detecting an analyte in a liquidsample, which process comprises forming a plurality of reagent dots on amatrix to make a test device comprising at least two of said reagentdots that do not overlap and are sufficiently spaced apart from eachother so that when said liquid sample flows laterally along said matrix,flow of said liquid sample to, through and/or around one of said tworeagent dots does not substantially affect flow of said liquid sampleto, through and/or around said other reagent dot, wherein each of saidtwo reagent dots is neither a reagent line across the entire width ofsaid matrix in a direction perpendicular to the direction of said liquidsample flow nor a complete circle of a reagent line, and after a liquidsample flows laterally along said test device and passes said at leasttwo reagent dots, said at least two reagent dots form a predeterminedpattern to indicate presence, absence and/or amount of said analyte insaid liquid sample.

The plurality of reagent dots can form any suitable predeterminedpattern. In some embodiments, the plurality of reagent dots form a line,multiple lines, a symbol, a geometric shape or an alpha-numeric shape.Any suitable alpha-numeric shape can be formed. Exemplary alpha-numericshapes include a letter, a word, a number or a combination thereof. Thereagent dot line can be formed at any suitable direction. In oneexample, the line is substantially parallel to the liquid sample flowdirection. In another example, the line is substantially perpendicularto the liquid sample flow direction. In still another example, thereagent dots form multiple lines that comprise at least one line that issubstantially parallel to the liquid sample flow direction and at leastone line that is substantially perpendicular to the liquid sample flowdirection.

The reagent dots can be formed on the matrix by any suitable methods. Insome embodiments, the plurality of reagent dots is formed by dispensinga reagent at predetermined locations on the matrix. Any suitabledispensing techniques or methods can be used. For example, the reagentcan be dispensed by a drop on demand method or a printing process usinga mechanical transfer of the reagent. Any suitable drop on demandmethods can be used. For example, the drop on demand method can beconducted using an inkjet or solenoid valve based dispenser, apiezoelectric dispenser, screen printing, airjet or airbrush technology,hollow pin printing, or near-contact dispensing.

The reagent dots in the test device can have any suitable size(s). Inone example, at least one of the reagent dots has a diameter of about0.1-1 um, 1-10 um, 10-50 um, 51-100 um, 101-200 um, 201-300 um, 301-400um, 401-500 um and 501-1000 um. In another example, at least a quarter,a third, half or all reagent dots have a diameter of about 0.1-1 um,1-10 um, 10-50 um, 51-100 um, 101-200 um, 201-300 um, 301-400 um,401-500 um or 501-1000 um. In still another example, at least one of thereagent dots has a diameter or surface area that is about 10%, 5%, 1%,0.5%, 0.1%, 0.05%, 0.01%, 0.001% or smaller diameter or surface area ofthe length, width or surface area of the matrix calculated by the widthand length of the membrane. In yet another example, at least a quarter,a third, half or all reagent dots have a diameter or surface area thatis about 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or smallerdiameter or surface area of the length, width or surface of the matrix.

The reagent dots in the test device can have the same or differentsize(s) or diameter(s). In one example, at least a quarter, a third,half or all reagent dots have substantially the same size or diameter.In another example, at least a quarter, a third, half or all reagentdots have substantially different sizes or diameters.

The reagent dots in the test device can have any suitable shapes, e.g.,any suitable regular or irregular shape. In one example, at least one ofthe reagent dots has a shape that is a line, a circle, a rod, a square,a triangle, a rectangle or an irregular shape. In another example, atleast a quarter, a third, half or all reagent dots have a shape that isa line, a circle, a rod, a square, a triangle, a rectangle or anirregular shape. The reagent dots in the test device can have the sameor different shape(s). In one example, at least a quarter, a third, halfor all reagent dots have the same shape. In another example, at least aquarter, a third, half or all reagent dots have different shapes.

The reagent dots can have any suitable space(s) or distance(s) betweenor among the dots. In one example, the distance between or among thereagent dots is about 1-10 um, 10-50 um, 51-100 um, 101-200 um, 201-300um, 301-400, 401-500, or 501-600 um. The space(s) or distance(s) betweenor among the reagent dots can be the same or different. In one example,the space or distance between or among at least a quarter, a third, halfor all reagent dots is substantially the same. In another example, thespace or distances between or among at least a quarter, a third, half orall reagent dots are different.

The reagent dots can be formed on any suitable places or side(s) of thematrix. In one example, the present process comprises forming a singlelayer or multiple layers of the plurality of reagent dots. In anotherexample, the present process comprises forming at least a layer of theplurality of reagent dots on both sides of the matrix. In still anotherexample, the present process comprises forming multiple layers of theplurality of reagent dots on both sides of the matrix. In yet anotherexample, the present process further comprises a drying step betweenforming the multiple layers of the plurality of reagent dots.

In some embodiments, the present invention provides for a test devicefor detecting an analyte in a liquid sample, which is manufactured bythe above process(es).

E. Exemplary Embodiments

In some embodiments, the present invention provides for a method for thedispensing of capture reagent geometries in lateral flow formats thatallows for the generation of signals in non standard formats, includingdots (or “pixels”) which do not perturb the flow of reagents in the testmatrix with the result that multiple small signal features can bedeveloped and may be combined to form symbols and letters. In otherembodiments, the present invention provides for a new device, a lateralflow test with configurable indicia compatible with multiplexing andquantitative testing. Embodiments of novel and improved assay formatsthat can be generated as a result of the application of this method,unique device constructs that enable multiplex testing to be performedin a single device, and unique device constructs that enable improvedquantitative and qualitative interpretation of test results are alsoprovided.

One aspect of the invention is directed to a method for the dispensingof chemical or biological reagents (“reagents”) onto porous and/or solidmatrices such as plastic films in predetermined “pixilated” patternsthat have low resistance to the flow of fluids and particulates throughthe printed reagents. These patterns have dimensional features that arespatially separated at distances on the order of the diffusion distanceof the sample/conjugate flow normal to the principal direction of flowin the system. The dimensions of these features and positioning relativeto one another ensures that each feature can interact with the reagentsin the test system individually without occluding the reaction matrix orotherwise causing disruption in flow. These feature dimensions andrelative positions will vary with material and reagent characteristicssuch as the surface type (solid/porous), pore size if porous, andcapture reagent affinity.

The method can be used to generate complex or simple reagent patterns ona lateral flow membrane or other matrix that develop not onlyperpendicular to the direction of flow as in a traditional lateral flowsystem, but parallel to the direction of flow. This method can also beused to generate such features on a variety of matrices that can conductfluids, including nitrocellulose membranes, glass fibers and plasticsand films. This method can further be used to generate a variety ofsymbols, including lines, dots and geometric and alpha-numeric shapes.

In certain implementations of the invention, the method may be used forproducing quantitative tests. The pixel format provides for the abilityto detect concentration depletion of an analyte as it flows through afield of pixels. This is shown schematically in FIGS. 6( a) and 6(b).The same principle applies to both particulate and non-particulate(molecular) labels. The exemplary test format is as follows: 1. dispensea pixel field across the lateral test strip membrane where the pixelfield consists of a series of reagent dots across the width of themembrane in patterns such as those shown. The dots may be offset orphysically separated at such a distance that lateral diffusion can occurbetween spots. When sample flows through this field, analyte will bindto the dots and become depleted as it migrates further up the membrane.When conjugate is subsequently flowed through the system, a depletion ofanalyte concentration is illustrated by decreasing signal intensity frombottom to top of the matrix. This can form the basis of a test formatfor achieving titration or quantification of the analyte through adirect measurement of the concentration gradient either by eye or usinga reader.

A further refinement on this technique can be envisaged by titrating thecapture reagent in one dimension, leading to the formation of a2-dimensional binding array when sample is run up the strip. (Shownschematically in FIG. 7). In this system, side by side lines of bindingpixels are dispensed in the direction of flow, each line containingbinding reagent at a different concentration. The two dimensionalbinding pattern thus created yields a system that can produce aquantitative measure of analyte in the system that has a broad dynamicrange

A further refinement involves the use of this format for multiplexeddetection or quantification. An example of how the pixel format can beused for multiplexed formats is shown in FIG. 8, which shows the pixelpattern for 4 analytes using a format of 4×20 pixels with a pixel sizeof about 250 um with 100 um separation between pixels. The 20 pixeldimension is laid out in the sample flow direction.

This method can be used to produce alpha-numeric or graphical resultssince signal generation is not dependent on orienting the capturereagent perpendicular to the direction of flow. Multiple potentialembodiments can be envisaged, with the generation of results in the formof words or symbols for easy interpretation, or the creation of codednumerical results or identifiers on the product.

In the present embodiment the production of the assay matrix isdispensing of individual drops. The method for dispensing of the arrayscan be based on a variety of technologies, including inkjet, BioJet,hollow pin or piezo-electric. Other dispensing methods may be envisaged.

In one preferred embodiment the dispensing technique is printing. Inthis embodiment the method involves printing of the reagent pixels inthe flow path to create individual features that are of a dimension (inall three axes) that allows each individual feature to capture analyteand signal reagent and develop completely, irrespective of the bindingaffinity of the reagent, as long as the reagent has some affinity forthe analyte.

The three dimensional matrix arrangement of the individual featuresgenerates larger patterns which, when viewed in or on a plane produceindicia representative of the test results such as symbols(alpha-numeric, lines or dots) that can be used for test interpretation.Result interpretation can be qualitative or quantitative and may beperformed by eye or by a reader. For qualitative tests, the presence orabsence of such indicia will be representative of the test result. Inthe instances where test results are quantitative in nature thequantity, size, or intensity of indicia may be the representativemeasure.

In another preferred embodiment the dispensing technique is multi-layerprinting. In this embodiment the method involves printing of thereagents in multiple layers in the flow path to create individualfeatures that are of a dimension (in all three axes) that allows eachindividual feature to capture analyte and signal reagent and developcompletely, irrespective of the binding affinity of the reagent, as longas the reagent has some affinity for the analyte. The multiple layers ofprinting can occur on the same side of the matrix or on opposite sidesof the matrix to create features at different depths within the matrix.

The three dimensional matrix arrangement of the individual featuresgenerates larger features which, when viewed in or on a plane produceindicia representative of the test results such as symbols(alpha-numeric, lines or dots) that can be used for test interpretation.Result interpretation can be qualitative or quantitative and may beperformed by eye or by a reader. For qualitative tests, the presence orabsence of such indicia will be representative of the test result. Inthe instances where test results are quantitative in nature thequantity, size, or intensity of indicia may be the representativemeasure.

Examples of Potential Embodiments

In all of the described embodiments, the signal reagents used togenerate the signal in the test would include visible, paramagnetic,optically excited particles or molecules. Optically excited wouldinclude fluorescence, luminescence, up converting phosphors) and alsoadd enzymes for visual, fluorescent and electrochemical signaling. Othertypes of signal generation could be envisaged.

1. Alpha Numeric Symbol on a Lateral Flow Strip

1.1. Exemplary Application: Pregnancy test with a + and − indicatingpositive or negative. The particular application is non-limiting and isfor illustration only. This example illustrates the method forgenerating a true + and − on a lateral flow strip.

In most pregnancy tests, two antibodies are used to bind to the hCGmolecule to create a positive signal. Anti hCG alpha can be striped onthe test line, and anti hCG beta is conjugated to the label used togenerate the signal. These are often monoclonal antibodies. The controlline on the system can therefore be an anti mouse antibody, which willbind directly to the conjugated anti hCG beta antibody whether hCG ispresent or not. In a typical system, the test line and control line aretwo independent lines, striped perpendicular to the direction of flow.In this embodiment, one line is striped perpendicular to flow, and theother is striped in the direction of flow, crossing each other in thecenter of the strip. When no analyte is present, only one line willdevelop, creating a “−”. When the analyte is present, a “+” will appear,when both lines develop. This is possible in our embodiment, while notpossible in standard lateral flow systems due to the fact that it ispossible to develop distinct lines in the direction of flow in oursystem. This format will also function when the lines are at an angle toflow, creating an “x” rather than a “+”.

1.2. This embodiment will also function in the situation where the assayis a competitive assay, such as a drugs of abuse test. In this instance,the presence of the analyte prevents the second line from forming.However the positive in this system would create a “−” symbol, so analternative symbol may be used (see Embodiment 2 below).

As well as a “+” or “−”, this method can be used to create words asreporting mechanisms in lateral flow. The word “Yes” can be printed onthe strips and develop in the presence of the analyte. In a biowarfareapplication such as an anthrax test, where interpretation of results iscritical, the word “Danger” could, for example appear, removing doubtfrom the warfighter or technician that a likely exposure event is inprogress.

2. Alpha Numeric Positive Reporting System in a Competitive Lateral FlowAssay

In competitive assay systems, the presence of the analyte results in thedisappearance of the test line. This format of reporting can lead toconfusion in the hands of some users, who are more used to theappearance of a line in the presence of a positive. Competitive assaysare important formats for the detection of small molecules, for exampledrugs of abuse, or many biomolecules of importance. The system ofgenerating symbols on a lateral flow strip can allow for the improvementof interpretation. For example, the control line could be configured asa “+” symbol, designed to develop in all cases. The test line could beprinted as an alphanumeric series, for example the word “Not”. Thus inthe case where no analyte is present, the result “Not +” appears. Whenthe analyte is present, the word “Not” will not develop and only the “+”symbol will develop. Other forms of this feature can be readilyenvisaged.

3. Quantification

3.1. One embodiment of this test format is to create a series ofindividual dots of capture reagent, or “Pixels” throughout the read areaof the test strip. As the sample moves through the read area, analytewill be bound at all of those capture dots. The analyte will thus bedepleted as the sample migrates through the system, potentiallyresulting in the creation of a gradient of spot intensity along thelength of the strip. This could be used to create a “Thermometer” styleresult, wherein the height and intensity of the developed area in thesystem can be related to the concentration of analyte in the system.This could be used for titration of analytes (e.g., cardiac biomarkerssuch as TnI or pro BNP, FABP or CKMB, or antibody concentrations) inquantitative or semi quantitative systems.

3.2. In a second embodiment, individual channels of printed reagents aregenerated on the membrane. The “channels” are discrete areas oriented inthe direction of flow, each consisting of a set of capture pixels asdescribed in the above example. There may be two or more discretechannels side by side in the direction of flow. No physical separationis required. The capture reagents may be different reagents, in theexample of a multiplexed test, such that each channel detects adifferent analyte in a single sample. The capture reagent may also bethe same reagent, however at different concentrations. Thus, thetitration effect described in 3.1 above can be performed in twodimensions, with a titration of the capture reagent in the axisperpendicular to flow, and a titration of the analyte occurring in thedirection of flow as it is depleted from the sample as the sample movesthrough the capture and read area. This will allow for titrations andquantification of analyte over a very broad dynamic range. This may beuseful in quantifying levels of an analyte in a sample that can bepresent over a broad range in normal and test subjects or samples (e.g.hCG where applications exist for detecting levels as low as 10 mIU/mland as high as 250,000 mIU/ml, a dynamic range that cannot be handled bymost lateral flow systems).

This format may also be useful in assessing binding kinetics andscreening for antibodies or other reagents for use in lateral flow orother assay formats. For example, when screening for antibodies forlateral flow formats, it is useful to screen for antibodies with highaffinity. Those antibodies will produce distinctive binding features ina pixilated assay (e.g., high affinity binding will typically develop asa strong leading edge of a dot rather than an evenly developed dot). Thehigher the affinity, the more of the antibody will be quickly pulled outof the sample as it migrates up the strip. A high affinity bindingsystem could therefore be indicated by evaluating the pattern ofdevelopment of individual dots, as well as observing how high up thestrip the development pattern goes.

4. Test Identification

Printing of alpha numeric information on strips that will only appearwhen the strip is developed. This code could develop using the controlline binding system, and may in fact be the control line or may bevisible elsewhere on the device. This can be used for anticounterfeiting, for QC or product identification purposes, or inapplications that require verification of the strip identity (e.g.,telephone or internet based diagnostics linked to prescription).

5. Non-Linear Embodiments

The pixilation concept allows for the creation of alternate assayformats, such as radial systems, where sample is added to a central welland migrates in all directions around the well. This could be used formultiplexing or for quantification as described in 3 above. Thepixilation concept may also be used to create results in complex flowpatterns such as in printed liquidic circuits or other microfluidicformats.

6. Flow Through or Hybrid Flow Through/Lateral Flow Systems

This pixilation concept may be applied in flow through assay formats,where there is some form of vertical movement of fluids as well ashorizontal movement. Flow through assays, where reagents move verticallythrough an assay as against horizontally, are typically used wheremultiple steps are not an issue (for example in the clinical laboratorywhere trained staff are operating the tests and a CLIA waiver is notrequired). They are also used in low cost, low complexity environments,such as for HIV testing in the developing world. One of the issuestypically encountered with the flow through format is interpretation ofresults, which involves the interpretation of individual dots by eye oroccasionally by reader. This system could be used to generate patternsof dots that are easier to interpret.

7. Non Standard Materials and Configurations

The pixilation concept or format lends itself to application inalternative assay configurations. In essence, this method of preparationof reagent patterns on membranes allows for the creation of arrays inlow cost membranes such as nitrocellulose, which can then be probedusing a flow of reagents laterally through the membrane. This can beapplied to papers or other matrices. This methodology can also becoupled with tethering methods such as Quantiscientifics' proteintethering system to create easy to use two dimensional arrays that canbe probed using a lateral flow technique, allowing for ease of use,removal of the need for complex and expensive processing units andexpensive plates or slides. This may also be applicable to nucleic acidarrays.

8. Three Dimensional Pixel Arrays

In this embodiment, an array of reagents is printed on each side of aporous, hydrophilic matrix such as nitrocellulose or a solidhydrophilized matrix such as a plastic that has been plasma treated orhydrophilized in another way. Those capture reagents may be directedagainst the same or different analytes. When the test is run the assaydevelops on both sides of the matrix and can be individually analyzed.

9. Signal Readout

Any or all of these embodiments are intended to be interpreted either byeye or by a reader system.

In one aspect, the present invention provides for a diagnostic testdevice having an array of one or more reagents dispensed in a pixelatedarray on a single strip interspaced in a manner that does not impedefluidic flow across the substrate thereby allowing continuous reactionsbetween the sample and various reagents to occur uniformly across andalong the test strip. The individual pixels of reagents can react withthe specific elements of the test sample to collectively or individuallyform signals which can be interpreted quantitatively or qualitatively.The signal can be a predetermined indicia shape.

In some embodiments, two or more differing reagent sets can be dispensedsequentially onto a single strip in order to produce detectible signalsindicative of two or more agents. The two or more differing reagent setscan be dispensed concurrently in a single array onto a single strip inorder to produce detectible signals indicative of two or more agents inthe test sample. The two or more reagents can also be dispensed in a 3dimensional array, having at least two layers of reagent beingsuperimposed. In other embodiments, the reagent sets can be dispensed ina pattern allowing for the development of signals in the direction offlow of the strip, allowing for development of qualitative orquantitative, single or multiplexed results.

In another aspect, the present invention provides for a method formanufacturing a diagnostic test device having low flow resistance toliquidized test sample media (material) in the analytical matrixincluding the steps of: dispensing at least one reagent material in aprescribed 2 dimensional pixelated array pattern onto a hydrophilic orhydrophilized substrate material, each of the pixels having vacuousfluid flow volumes there between such that each pixel will developwithout creating significant resistance in the flow path of the device.

In some embodiments, reagent can be dispensed in a manner that reactionsbetween the sample material and specific reagent pixels produces atleast one detectable signal on the substrate material such as anidentifiable symbol or prescribed pattern indicative of the test result.Indicia can be alpha-numeric, geometric or other predetermined shapes.Exemplary reagents include antibodies, antigens, nucleic acid, e.g., DNAor RNA, based molecules, proteins, peptides or any other large or smallmolecules.

The pixel can have any suitable size. In some embodiments, the pixel canhave a size or diameter at about 0.1-1 um, 1-10 um, 10-50 um, 51-100 um,101-200 um, 201-300 um, 301-400 um, 401-500 um, or 501-1000 um. Thepixels can have any suitable spacing between or among the pixels,especially the adjacent pixels. In some embodiments, the pixels oradjacent pixels can have a minimum spacing or distance of about 1-10 um,10-50 um, 51-100 um, 101-200 um, 201-300 um, 301-400, 401-500, or501-600 um.

Any suitable method or technique can be used to dispense the pixels. Insome embodiments, the pixel dispensing method can be drop on demandusing inkjet or solenoid valve based dispensers, piezoelectricdispensers, screen printing, airjet or airbrush technology, hollow pinprinting, near-contact dispensing and any other commercial printingprocess using a mechanical transfer of reagents.

In still another aspect, the present invention provides for a method ofmanufacturing a 3 dimensional reaction matrix in a diagnostic test by:dispensing reagent in a prescribed pixelated pattern, drying thedispensed reagent, dispensing at least one additional layer of reagentpixels on top of the first dispensed reagent pattern layer or on theopposite side of the porous matrix, creating a three dimensional pixelstructure within the matrix, drying each layer after dispensing.

In yet another aspect, the present invention provides for a method ofmanufacturing a diagnostic test that includes a reaction matrix that hasbeen processed using a method that includes the steps of: dispensing ortransferring of reagent in a prescribed pixelated pattern, drying thedispensed reagent, dispensing or transfer of at least one additionallayer of reagent pixels on top of the first dispensed reagent patternlayer or on the opposite side of a non porous matrix, drying each layerafter dispensing, thereby creating a matrix with a pixilated flowpath oneither side of the matrix allowing for two tests to be runsimultaneously on the same matrix.

The present invention is further illustrated by the following exemplaryembodiments:

1. A test device for detecting an analyte in a liquid sample, whichdevice comprises a plurality of reagent dots on a matrix, wherein atleast two of said reagent dots do not overlap and are sufficientlyspaced apart from each other so that when said liquid sample flowslaterally along said matrix, flow of said liquid sample to, throughand/or around one of said two reagent dots does not substantially affectflow of said liquid sample to, through and/or around said other reagentdot, each of said two reagent dots is neither a reagent line across theentire width of said matrix in a direction perpendicular to thedirection of said liquid sample flow nor a complete circle of a reagentline, and after a liquid sample flows laterally along said test deviceand passes said at least two reagent dots, said at least two reagentdots form a predetermined pattern to indicate presence, absence and/oramount of said analyte in said liquid sample.

2. The test device of embodiment 1, wherein the plurality of reagentdots comprises two reagent dots.

3. The test device of embodiment 1, wherein the plurality of reagentdots comprises more than two reagent dots.

4. The test device of embodiment 3, wherein the plurality of reagentdots comprises at least 10, 50, 100, 500, 1,000, 5,000, 10,000 or morereagent dots.

5. The test device of any of the embodiments 3-4, wherein at least aquarter, a third, half or all reagent dots do not overlap and aresufficiently spaced apart from each other so that when the liquid sampleflows laterally along the matrix, flow of the liquid sample to, throughand/or around one of the reagent dots does not substantially affect flowof the liquid sample to, through and/or around the other reagent dots.

6. The test device of embodiment 1, wherein the predetermined pattern isselected from the group consisting of a line, multiple lines, a symbol,a geometric shape and an alpha-numeric shape.

7. The test device of embodiment 6, wherein the alpha-numeric shape is aletter, a word, a number or a combination thereof.

8. The test device of embodiment 6, wherein the line is substantiallyparallel to the liquid sample flow direction.

9. The test device of embodiment 6, wherein the line is substantiallyperpendicular to the liquid sample flow direction.

10. The test device of embodiment 6, wherein the multiple linescomprises at least a line that is substantially parallel to the liquidsample flow direction and at least a line that is substantiallyperpendicular to the liquid sample flow direction.

11. The test device of any of the embodiments 1-10, wherein theplurality of reagent dots comprises different reagents and the testdevice is used to detect multiple analyte in the liquid sample.

12. The test device of any of the embodiments 1-10, wherein theplurality of reagent dots comprises the same reagent and the test deviceis used to detect the amount of the analyte in the liquid sample.

13. The test device of the embodiment 12, wherein the plurality ofreagent dots comprises the same amount of the reagent.

14. The test device of the embodiment 12, wherein the plurality ofreagent dots comprises the different amounts of the reagent.

15. The test device of any of the embodiments 1-14, wherein at least oneof the reagent dots has a diameter of about 0.1-1 um, 1-10 um, 10-50 um,51-100 um, 101-200 um, 201-300 um, 301-400 um, 401-500 um and 501-1000um, or at least one of the reagent dots has a diameter or surface areathat is about 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or smallerdiameter or surface area of the length, width or surface area of thematrix.

16. The test device of the embodiment 15, wherein at least a quarter, athird, half or all reagent dots have a diameter of about 0.1-1 um, 1-10um, 10-50 um, 51-100 um, 101-200 um, 201-300 um, 301-400 um, 401-500 umor 501-1000 um, or at least a quarter, a third, half or all reagent dotshave a diameter or surface area that is about 10%, 5%, 1%, 0.5%, 0.1%,0.05%, 0.01%, 0.001% or smaller diameter or surface area of the length,width or surface of the matrix.

17. The test device of the embodiment 15, wherein at least a quarter, athird, half or all reagent dots have substantially the same size ordiameter.

18. The test device of any of the embodiments 1-17, wherein at least oneof the reagent dots has a shape selected from the group consisting of aline, a circle, a rod, a square, a triangle, a rectangle and anirregular shape.

19. The test device of the embodiment 18, wherein at least a quarter, athird, half or all reagent dots have a shape selected from the groupconsisting of a line, a circle, a rod, a square, a triangle, a rectangleand an irregular shape.

20. The test device of the embodiment 18, wherein at least a quarter, athird, half or all reagent dots have the same shape.

21. The test device of any of the embodiments 1-20, wherein the distancebetween edges of at least two of the reagent dots is about 1-10 um,10-50 um, 51-100 um, 101-200 um, 201-300 um, 301-400, 401-500, or501-600 um.

22. The test device of the embodiment 21, wherein the distance betweenat least a quarter, a third, half or all reagent dots is substantiallythe same.

23. The test device of any of the embodiments 1-22, wherein at least oneof the reagent dots comprises a reagent that is capable of binding to ananalyte or another binding reagent that is capable of binding to ananalyte.

24. The test device of embodiment 23, wherein the reagent is capable ofspecifically binding to an analyte or another binding reagent that iscapable of binding to an analyte.

25. The test device of the embodiment 23, wherein at least a quarter, athird, half or all reagent dots comprise a reagent that is capable ofbinding to an analyte or another binding reagent that is capable ofbinding to an analyte.

26. The test device of embodiment 25, wherein the reagents are capableof specifically binding to an analyte or another binding reagent that iscapable of binding to an analyte.

27. The test device of any of the embodiments 1-26, which comprisesmultiple layers of the plurality of reagent dots.

28. The test device of any of the embodiments 1-27, which comprises atleast a layer of the plurality of reagent dots on both sides of thematrix.

29. The test device of any of the embodiments 1-27, wherein the reagentsare inorganic molecules, organic molecules or complexes thereof.

30. The test device of embodiment 25, wherein the organic molecules areselected from the group consisting of an amino acid, a peptide, aprotein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid,a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipidand a complex thereof.

31. The test device of embodiment 30, wherein the protein is an antigenor an antibody.

32. The test device of any of the embodiments 1-31, wherein the matrixhas a porous structure.

33. The test device of embodiment 32, wherein the matrix comprisesnitrocellulose, glass fiber, polypropylene, polyethylene (preferably ofvery high molecular weight), polyvinylidene flouride, ethylenevinylacetate, acrylonitrile and/or polytetrafluoro-ethylene.

34. The test device of any of the embodiments 1-31, wherein the matrixhas a non-porous structure.

35. The test device of embodiment 32, wherein the matrix comprises aplastics, a film of a matrix having a hydrophilic surface, or a materialwith a controlled contact angle with the sample liquid.

36. The test device of embodiment 1, wherein the plurality of reagentdots comprises the same binding reagent that is capable of binding to ananalyte or another binding reagent that is capable of binding to theanalyte, the plurality of reagent dots form a line that is substantiallyparallel to the liquid sample flow direction, as the liquid sample flowslaterally along the test device, the analyte, if present in the liquidsample, becomes sequentially bound to the binding reagent at each of thereagent dots until the analyte is depleted by binding to the upstreamreagent dot(s), the binding of the analyte to the reagent dot(s)generates a dateable signal at the reagent dot(s), and the intensityand/or the number of the dateable signal at the reagent dot(s) providesa quantitation or a semi-quantitation of the analyte in the liquidsample.

37. The test device of embodiment 1, wherein the plurality of reagentdots comprises different binding reagents that are capable of binding todifferent analytes or other binding reagents that are capable of bindingto the analytes, the plurality of reagent dots form a line that issubstantially parallel to the liquid sample flow direction, as theliquid sample flows laterally along the test device, the analytes, ifpresent in the liquid sample, become bound to the binding reagents ateach of the reagent dots, the binding of the analytes to the reagentdots generates dateable signals at the reagent dots, and the presenceand/or intensity of the dateable signals at the reagent dots indicatesthe presence and/or amount of the analytes in the liquid sample.

38. The test device of embodiment 1, wherein the plurality of reagentdots comprises different groups of binding reagents, each group of thebinding reagents is capable of binding to the same analyte or anotherbinding reagent that is capable of binding to the same analyte, and thebinding reagents in different groups are capable of binding to differentanalytes or other binding reagents that are capable of binding todifferent analytes,

each group of the reagent dots forms a line that is substantiallyparallel to the liquid sample flow direction, and the different linesformed by the different groups of the reagent dots are substantiallyparallel to each other,

as the liquid sample flows laterally alone the test device, theanalytes, if present in the liquid sample, become sequentially bound tothe binding reagents at each of the reagent dots in each group of thereagent dots until the analytes are depleted by binding to the upstreamreagent dots, the binding of the analytes to the reagent dots generatesdateable signals at the reagent dots, and the intensity and/or thenumber of the dateable signals at the reagent dots provides aquantitation or a semi-quantitation of the different analytes in theliquid sample.

39. The test device of embodiment 1, wherein the plurality of reagentdots comprises the same binding reagent that is capable of binding to ananalyte or another binding reagent that is capable of binding to theanalyte, the plurality of reagent dots form multiple lines that aresubstantially parallel to the liquid sample flow direction, the reagentdots in each line comprise the same amount of the binding reagent, butthe reagent dots in different lines comprise the different amounts ofthe binding reagent,

as the liquid sample flows laterally alone the test device, the analyte,if present in the liquid sample, becomes sequentially bound to thebinding reagent at each of the reagent dots in each of the lines untilthe analyte is depleted by binding to the upstream reagent dot(s) ineach of the lines, the binding of the analyte to the reagent dot(s)generates a dateable signal at the reagent dot(s), and the intensityand/or the number of the dateable signal at the reagent dot(s) providesa quantitation or a semi-quantitation of the analyte in the liquidsample.

40. The test device of embodiment 39, wherein from one end to the otherend of the test device, in the direction perpendicular to the directionof said liquid sample flow, the reagent dots in different lines comprisethe sequentially different amounts of the binding reagent.

41. The test device of embodiment 1, wherein the plurality of reagentdots comprises two different groups of binding reagents, one group ofthe reagent dots forms a line that is at a first angle relative to theliquid sample flow direction, and the other group of the reagent dotsforms a line that is at a second, different angle relative to the liquidsample flow direction,

after the liquid sample flows laterally along the test device, thereagent dots in one of the lines generate a signal indicating thepresence and/or amount of an analyte in the liquid sample, and thereagent dots in the other line generate a control signal indicating thetest is properly conducted, and

when the liquid sample comprises the analyte and the test is properlyconducted, the two lines of the reagent dots generate a positive symbol,indicating the presence and/or amount of the analyte in the liquidsample, and when the liquid sample does not comprise the analyte and thetest is properly conducted, only one line of the reagent dots generatesa negative symbol, indicating the absence of the analyte in the liquidsample.

42. The test device of embodiment 41, wherein one group of the reagentdots forms a line that is substantially parallel to the liquid sampleflow direction, and the other group of the reagent dots forms a linethat is substantially perpendicular to the liquid sample flow direction,and

when the liquid sample comprises the analyte and the test is properlyconducted, the two lines of the reagent dots generate a “+” symbol,indicating the presence and/or amount of the analyte in the liquidsample, and when the liquid sample does not comprise the analyte and thetest is properly conducted, only one line of the reagent dots generatesa “−” symbol, indicating the absence of the analyte in the liquidsample.

43. The test device of embodiment 1, wherein the plurality of reagentdots comprises two different groups of binding reagents, after theliquid sample flows laterally along the test device, reagent dots in onegroup generate an alpha-numeric signal indicating the presence and/oramount of an analyte in the liquid sample, and the reagent dots in theother group generate a control symbol signal indicating the test isproperly conducted.

44. The test device of embodiment 43, wherein the alpha-numeric signalis a word.

45. The test device of embodiment 44, wherein the word is yes, Pos,Positive, Neg, Negative, No, or OK.

46. The test device of any of the embodiments 43-45, wherein the controlsymbol signal is a “+” sign.

47. The test device of any of the embodiments 43-46, which is configuredfor a competitive test.

48. The test device of embodiment 1, wherein the plurality of reagentdots comprises a reagent that binds to an intended binder and thebinding pattern between the reagent and the binder formed on the reagentdots indicates a kinetic property of the binding between the reagent andthe binder.

49. The test device of embodiment 48, wherein the reagent is an antigen,the binder is an antibody to the antigen, and the binding patternbetween the antigen and the antibody formed on the reagent dotsindicates a kinetic property of the binding between the antigen and theantibody.

50. The test device of embodiment 49, wherein the kinetic propertycomprises the binding affinity of the binding between the antigen andthe antibody.

51. The test device of embodiment 50, wherein the binding pattern of aleading edge on the dot(s) indicates that the antibody has a highbinding affinity for the antigen.

52. The test device of any of the embodiments 1-51, which comprises atleast one group of the reagent dots that generate an additional signalthat is not related to the presence, absence and/or amount of theanalyte in the liquid sample, or whether the test is properly conducted.

53. The test device of embodiment 52, wherein the additional signalindicates the authenticity, quality and/or identification of the testdevice, or identification of the liquid sample.

54. The test device of any of the embodiments 52-53, wherein theadditional signal comprises an alpha-numeric signal.

55. The test device of embodiment 1, which comprises at least one groupof the reagent dots that form a circle around the sample applicationlocation, and the liquid sample moves radially to pass the group of thereagent dots.

56. The test device of embodiment 1, which further comprises a flowthrough device portion.

57. The test device of any of the embodiments 1-56, wherein the matrixis in the form a strip or a circle.

58. The test device of any of the embodiments 1-56, wherein the matrixis a single element or comprises multiple elements.

59. The test device of any of the embodiments 1-58, which furthercomprises a sample application element upstream from and in fluidcommunication with the matrix.

60. The test device of any of the embodiments 1-59, which furthercomprises a liquid absorption element downstream from and in fluidcommunication with the matrix.

61. The test device of any of the embodiments 1-60, which furthercomprises a control location comprising means for indicating proper flowof the liquid sample and/or a valid test result.

62. The test device of any of the embodiments 1-61, wherein at least aportion of the matrix is supported by a solid backing.

63. The test device of any of the embodiments 1-62, wherein a portion ofthe matrix, upstream from the at least two of the reagent dots,comprises a dried, labeled reagent, the labeled reagent being capable ofbeing moved by a liquid sample and/or a further liquid to the at leasttwo of the reagent dots and/or a control location to generate adetectable signal.

64. The test device of embodiment 63, wherein the dried, labeled reagentis located downstream from a sample application place on the testdevice.

65. The test device of embodiment 63, wherein the dried, labeled reagentis located upstream from a sample application place on the test device.

66. The test device of any of the embodiments 1-62, which furthercomprises, upstream from the at least two of the reagent dots, aconjugate element that comprises a dried, labeled reagent, the labeledreagent being capable of moved by a liquid sample and/or a furtherliquid to the at least two of the reagent dots and/or a control locationto generate a detectable signal.

67. The test device of embodiment 66, wherein the conjugate element islocated downstream from a sample application place on the test device.

68. The test device of embodiment 66, wherein the conjugate element islocated upstream from a sample application place on the test device.

69. The test device of any of the embodiments 63-68, wherein the labeledreagent binds to an analyte in the liquid sample.

70. The test device of any of the embodiments 63-68, wherein the labeledreagent competes with an analyte in the liquid sample for binding to abinding reagent for the analyte at the at least two of the reagent dots.

71. The test device of any of the embodiments 63-70, wherein the labelis a soluble label.

72. The test device of any of the embodiments 63-70, wherein the labelis a particle label.

73. The test device of any of the embodiments 63-72, wherein the labeledreagent is dried in the presence of a material that: a) stabilizes thelabeled reagent; b) facilitates solubilization or resuspension of thelabeled reagent in a liquid; and/or c) facilitates mobility of thelabeled reagent.

74. The test device of embodiment 73, wherein the material is selectedfrom the group consisting of a protein, a peptide, a polysaccharide, asugar, a polymer, a gelatin and a detergent.

75. The test device of any of the embodiments 1-74, wherein a sampleliquid alone is used to transport the analyte and/or the labeled reagentto the at least two of the reagent dots.

76. The test device of any of the embodiments 1-74, wherein a developingliquid is used to transport the analyte and/or the labeled reagent tothe at least two of the reagent dots.

77. The test device of any of the embodiments 1-76, which furthercomprises a housing that covers at least a portion of the test device,wherein the housing comprises a sample application port to allow sampleapplication upstream from or to the at least two of the reagent dots andan optic opening around the at least two of the reagent dots to allowsignal detection at the two of the reagent dots.

78. The test device of embodiment 77, wherein the housing covers theentire test device.

79. The test device of embodiment 77, wherein at least a portion of thesample receiving portion of the matrix or the sample application elementis not covered by the housing and a sample is applied to the portion ofthe sample receiving portion of the matrix or the sample applicationelement outside the housing and then transported to the at least two ofthe reagent dots.

80. The test device of any of the embodiments 77-79, wherein the housingcomprises a plastic material, a biodegradable material or a cellulosicmaterial.

81. The test device of any of the embodiments 1-80, wherein the liquidsample has moved laterally along the test device to generate detectablesignal(s) at the at least two of the reagent dots.

82. A method for detecting an analyte in a liquid sample, which methodcomprises:

a) contacting a liquid sample with the test device of any of theembodiments 1-80, wherein the liquid sample is applied to a site of thetest device upstream of the at least two of the reagent dots;

b) transporting an analyte, if present in the liquid sample, and alabeled reagent to the at least two of the reagent dots; and

c) assessing the presence, absence, amount and/or pattern of signal(s)generated by the labeled reagent at the at least two of the reagent dotsto determining the presence, absence and/or amount of the analyte in theliquid sample.

83. The method of embodiment 82, wherein the liquid sample and thelabeled reagent are premixed to form a mixture and the mixture isapplied to the test device.

84. The method of embodiment 82, wherein the test device comprises adried labeled reagent before use and the dried labeled reagent issolubilized or resuspended, and transported to the at least two of thereagent dots by the liquid sample.

85. The method of embodiment 84, wherein the dried labeled reagent islocated downstream from the sample application site, and the driedlabeled reagent is solubilized or resuspended, and transported to the atleast two of the reagent dots by the liquid sample.

86. The method of embodiment 84, wherein the dried labeled reagent islocated upstream from the sample application site, and the dried labeledreagent is solubilized or resuspended, and transported to the at leasttwo of the reagent dots by another liquid.

87. The method of embodiment 84, wherein the labeled reagent issolubilized or resuspended, and transported to the at least two of thereagent dots by the liquid sample alone.

88. The method of embodiment 84, wherein the analyte and/or labeledreagent is solubilized or resuspended, and transported to the at leasttwo of the reagent dots by another liquid.

89. The method of any of the embodiments 84-88, wherein the liquidsample is body fluid sample.

90. The method of embodiment 89, wherein the body fluid sample isselected from the group consisting of a whole blood, a serum, a plasmaand a urine sample.

91. The method of any of the embodiments 84-88, wherein the liquidsample is a sample derived from a biological, a forensics, a food, abiowarfare, or an environmental source.

92. The method of any of the embodiments 82-91, which is used toquantify or semi-quantify the amount of an analyte in a liquid sample.

93. The method of any of the embodiments 82-91, which is used to detectmultiple analytes in a liquid sample.

94. The method of embodiment 93, which is used to quantify orsemi-quantify the amounts of the multiple analytes in the liquid sample.

95. The method of any of the embodiments 82-94, wherein the analyte isselected from the group consisting of a cell, a virus and a molecule.

96. The method of any of the embodiments 82-94, wherein the analyte isselected from the group consisting of hCG, hLH, hFSH, hTSH, a cardiacbiomarker, an antigen of an infectious organism, an antibody to aninfectious organism and disease marker.

97. A process for manufacturing a test device for detecting an analytein a liquid sample, which process comprises forming a plurality ofreagent dots on a matrix to make a test device comprising at least twoof said reagent dots that do not overlap and are sufficiently spacedapart from each other so that when said liquid sample flows laterallyalong said matrix, flow of said liquid sample to, through and/or aroundone of said two reagent dots does not substantially affect flow of saidliquid sample to, through and/or around said other reagent dot, whereineach of said two reagent dots is neither a reagent line across theentire width of said matrix in a direction perpendicular to thedirection of said liquid sample flow nor a complete circle of a reagentline, and after a liquid sample flows laterally along said test deviceand passes said at least two reagent dots, said at least two reagentdots form a predetermined pattern to indicate presence, absence and/oramount of said analyte in said liquid sample.

98. The process of embodiment 97, wherein the plurality of reagent dotsform a predetermined pattern selected from the group consisting of aline, multiple lines, a symbol, a geometric shape and an alpha-numericshape.

99. The process of embodiment 98, wherein the alpha-numeric shape is aletter, a word, a number or a combination thereof.

100. The process of embodiment 98, wherein the line is substantiallyparallel to the liquid sample flow direction.

101. The process of embodiment 98 wherein the line is substantiallyperpendicular to the liquid sample flow direction.

102. The process of embodiment 98, wherein the multiple lines compriseat least one line that is substantially parallel to the liquid sampleflow direction and at least one line that is substantially perpendicularto the liquid sample flow direction.

103. The process of any of the embodiments 97-102, wherein the pluralityof reagent dots are formed by dispensing a reagent at predeterminedlocations on the matrix

104. The process of embodiment 103, wherein the reagent is dispensed bya drop on demand method or a printing process using a mechanicaltransfer of the reagent.

105. The process of embodiment 104, wherein the drop on demand method isconducted using an inkjet or solenoid valve based dispenser, apiezoelectric dispenser, screen printing, airjet or airbrush technology,hollow pin printing, and near-contact dispensing.

106. The process of any of the embodiments 97-105, wherein at least oneof the reagent dots has a diameter of about 0.1-1 um, 1-10 um, 10-50 um,51-100 um, 101-200 um, 201-300 um, 301-400 um, 401-500 um and 501-1000um.

107. The process of any of the embodiments 97-105, wherein at least oneof the reagent dots has a diameter or surface area that is about 10%,5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or smaller than the length,width or surface area of the matrix.

108. The process of any of the embodiments 97-107, wherein the distancebetween at least two of the reagent dots is about 1-10 um, 10-50 um,51-100 um, 101-200 um, 201-300 um, 301-400, 401-500, or 501-600 um.

109. The process of any of the embodiments 97-108, which comprisesforming multiple layers of the plurality of reagent dots.

110. The process of any of the embodiments 97-109, which comprisesforming at least a layer of the plurality of reagent dots on both sidesof the matrix.

111. The process of any of the embodiments 108-110, which furthercomprises a drying step between forming the multiple layers of theplurality of reagent dots.

112. A test device for detecting an analyte in a liquid sample, which ismanufactured by a process of any of the embodiments 97-111.

The ordinarily skilled artisan can appreciate that the present inventioncan incorporate any number of the preferred features described above.

Further features and advantages of the present invention will becomeapparent to those of skill in the art in view of the detaileddescription of preferred embodiments which follows, when consideredtogether with the attached drawings and claims.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims.

F. EXAMPLES Example 1

This experiment was conducted to generate a data set to demonstrateaspects of the “pixel” concept for signal development in lateral flowassays. Nitrocellulose membranes (CN 95, Sartorius) were dotted withvarious patterns and spot sizes as shown in FIGS. 10-16 using a Scienionaspirate-and-dispense piezoelectric-valve system. Biotin—BSA at aconcentration of 0.25 mg/ml (DCN) was used as the capture reagent on themembrane. A streptavidin-gold conjugate (40 nm gold, OD10, DCN) was usedas the signal reagent. Strips were developed by flowing thestreptavidin-gold conjugate laterally along the nitrocellulose membranein half strip format. The programmed symbols are shown in the A part ofFIGS. 10-16 and the symbols obtained from the actual tests are shown inthe B part of FIGS. 10-16.

Example 2

This experiment was conducted to illustrate that signal clarity can beimproved by removing background binding using a variety of membraneblocking buffers and running diluents and titration of the goldconjugate concentration. The various test strip configurations and testconditions are shown the in the following Table 1 and 2.

TABLE 2 Test Strip Configurations A 700 pL 35 × 35 200 cc B 10 spots 7nL C 700 pL 35 × 35 200 cc D 15 spots 101.5 nL E 700 pL 35 × 35 200 cc F5 spots 3.5 nL G 700 pL Double X 17 × 17 H 15 spots I 700 pL Double X 17× 17 J 10 spots K 700 pL DoubleX 17 × 17 L 5 spots M 700 pL Plus SignZig Zag N 20 spots O 700 pL Plus Sign 17 × 17 P 20 spots Q 700 pL PlusSign 35 × 35 R 20 spots S 700 pL DoubleX 17 × 17 20 spot

TABLE 3 Test Conditions A 100 uL PBS(+) 25 uL Conjugate 6 mm Strip, 10min runtime B 100 uL PBS(+) 10 uL Conjugate 6 mm Strip, 10 min runtime C100 uL 1XPBS w/0.1% Tween-20, 25 uL Conjugate, 10 min runtime D 100 uL1XPBS w/0.1% Tween-20 10 uL Conjugate 10 min runtime E 100 uL 1XPBSw/0.2% Tween-20, 10 uL conjugate, 10 min runtime F 100 uL 1XPBS w/0.2%Tween-20 + 0.01% BSA, 10 uL conjugate 7 min runtime K 100 uL 1XPBSw/0.2% Tween-20 + 0.05% BSA, 10 uL Conjugate, 7 min runtime M 100 uL1XPBS w/0.3% Tween-20 + 0.05% BSA, 10 uL Conjugate, 7 min runtime N 100uL 1XPBS w/0.2% Tween-20, 25 uL Conjugate, 10 min runtime, DCN membraneblocking buffer O 100 uL 1XPBS w/0.3% Tween-20 + 0.05% BSA, 20 uLConjugate, 7 min runtime P 100 uL 1XPBS, 25 uL conjugate, 10 minruntime, DCN membrane + conjugate pad blocking buffers Q 100 uL 1XPBSw/0.3% Tween-20 + 0.05% BSA, 15 uL Conjugate, 7 min runtime R 100 uL1XPBS w/0.3% Tween-20 + 0.05% BSA, 10 uL Conjugate, 7 min runtime S 100uL 1XPBS, 25 uL conjugate, 10 min runtime, DCN membrane blocking buffer

As shown in FIG. 17, background signal on the membrane was significantlydecreased with less gold conjugate. The “X” membranes were developedcompletely with 10 ul conjugate, and resulted in minimal backgrounddiscoloration (See Assay K). The “+” configurations required at least 25uL, thus producing dark pink membranes. Membrane blocking did not reducethe discoloration (see Assay N). The DCN conjugate pad blocking bufferdid not eliminate the background (see Assay S). In this experiment,optimal configurations appear to be K and S.

Example 3

This experiment was conducted to illustrate the use of a fluorescentlabel, e.g., Europium. Strips spotted with BSA-Biotin capture reagentwere developed using a Europium latex-Streptavidin conjugate (100 nmparticles, conjugate produced by DCN). In this experiment, 100 ul 1×PBS,25 uL (1:10 latex diluent) Eu latex conjugate in DCN conjugate padblocking buffer (10 mM Borate, 3% BSA, 1% PVP40, 0.25% Triton X 100, pH8.0) laterally along the nitrocellulose membrane. Results were observedand photographed under a black light at the 10 min runtime. As shown inFIG. 18, the Eu-latex conjugate developed a clearly visible signal withminimal background. This demonstrates the utility of the system withfluorescent labels and particles larger than the gold particles.

Example 4 Optimizing Signal Development Using the “Pixel” Concept forLateral Flow Assays

Nitrocellulose membranes (CN 95, Sartorius) were dotted (5 mL) withvarious patterns as shown in FIGS. 19A-19E using a BioDotaspirate-and-dispense microsolenoid-valve (“BioJet Plus”) system. Thecapture reagent on the membrane was anti-hCG antibody at a concentrationof 1 mg/ml. An anti-hCG antibody/gold conjugate (40 nm gold, OD10, DCN)was used as the signal reagent. The hCG analyte was detected at aconcentration of 1 UI/mL and diluted in 0.1% Tween-20 in 1×PBS. Stripswere developed in half strip format using liquid gold conjugate.

As shown in FIGS. 19A-19E, the vertical alignment of the spots preventedthe conjugate from moving up the strip uniformly. The outer edges of thetest lines were darkest and demonstrated that the fluid flow wasdeflected around the dispensed spots instead of flowing through or overthem. Variations in the spot pattern, such as increased length of thetest line and zig-zag patterns, did not enhance the results. Increasedspacing between the drops showed significant improvements to signaldevelopment.

Example 5 Applications of the “Pixel” Concept in Multiplexed LateralFlow Assays

Nitrocellulose membranes (CN 95, Sartorius) were dotted (5 mL) withvarious patterns as shown in FIGS. 20A-20D using a BioDotaspirate-and-dispense microsolenoid-valve (“BioJet Plus”) system. Thetest reagents on the membrane were anti-hCG antibody (1 mg/ml), andanti-myoglobin antibody (0.5 mg/ml). An anti-Mouse antibody capturereagent was used at a concentration of 0.5 mg/ml as a control for thesystem. An anti-hCG antibody/gold conjugate (40 nm gold, OD10, DCN) andan anti-Myoglobin antibody/gold conjugate (40 nm, OD10, DCN) were usedas the signal reagents. Strips were developed in half strip format usingliquid gold conjugate.

As shown in FIGS. 20A-20D, the assay accurately detected the variousconcentrations of the target analyte in the sample. The anti-Mouse,antimyoglobin and anti-hCG assays ran independently with no evidence ofcross-reactivity.

Example 6 Improving Construction of Various Spot Patterns Using aSmaller Dispense Volume

Nitrocellulose membranes (CN 95, Sartorius) were dotted (1 mL) withvarious patterns as shown in FIGS. 21A-21D using a BioDot Piezo dispensesystem. The test reagent on the membrane was anti-hCG antibody at aconcentration of 1 mg/ml. An anti-Mouse antibody capture reagent wasused at a concentration of 0.5 mg/ml as a control for the system. Ananti-hCG antibody/gold conjugate (40 nm gold, OD10, DCN) was used as thesignal reagent. The hCG analyte was detected at a concentration of 1UI/mL and diluted in 0.1% Tween-20 in 1×PBS. Strips were developed inhalf strip format using liquid gold conjugate

As shown in FIGS. 21A-21D, the configurations produced clean images witheven distribution of conjugate, regardless of line placement. Thesignals in FIGS. 21C and 21D were generated using hCG as analyte. Theflow was uninterrupted and able to produce apparent positive andnegative result illustrations (although no negative sample was run inthe tests shown in FIGS. 21C and 21D).

Citation of the above publications or documents is not intended as anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents.

1. A test device for detecting an analyte in a liquid sample, whichdevice comprises a plurality of reagent dots on a matrix, wherein atleast two of said reagent dots do not overlap and are sufficientlyspaced apart from each other so that when said liquid sample flowslaterally along said matrix, flow of said liquid sample to, throughand/or around one of said two reagent dots does not substantially affectflow of said liquid sample to, through and/or around said other reagentdot, each of said two reagent dots is neither a reagent line across theentire width of said matrix in a direction perpendicular to thedirection of said liquid sample flow nor a complete circle of a reagentline, and after a liquid sample flows laterally along said test deviceand passes said at least two reagent dots, said at least two reagentdots form a predetermined pattern to indicate presence, absence and/oramount of said analyte in said liquid sample, wherein the plurality ofreagent dots comprises the same binding reagent that is capable ofbinding to an analyte or another binding reagent that is capable ofbinding to the analyte, the plurality of reagent dots form a line thatis substantially parallel to the liquid sample flow direction, as theliquid sample flows laterally along the test device, the analyte, ifpresent in the liquid sample, becomes sequentially bound to the bindingreagent at each of the reagent dots until the analyte is depleted bybinding to the upstream reagent dot(s), the binding of the analyte tothe reagent dot(s) generates a dateable signal at the reagent dot(s),and the intensity and/or the number of the dateable signal at thereagent dot(s) provides a quantitation or a semi-quantitation of theanalyte in the liquid sample.
 2. The test device of claim 1, wherein theplurality of reagent dots comprises at least 10, 50, 100, 500, 1,000,5,000, 10,000 or more reagent dots.
 3. The test device of claim 1,wherein at least a quarter, a third, half or all reagent dots do notoverlap and are sufficiently spaced apart from each other so that whenthe liquid sample flows laterally along the matrix, flow of the liquidsample to, through and/or around one of the reagent dots does notsubstantially affect flow of the liquid sample to, through and/or aroundthe other reagent dots.
 4. The test device of claim 1, wherein at leasta quarter, a third, half or all reagent dots have a diameter of about0.1-1 um, 1-10 um, 10-50 um, 51-100 um, 101-200 um, 201-300 um, 301-400um, 401-500 um or 501-1000 um, or at least a quarter, a third, half orall reagent dots have a diameter or surface area that is about 10%, 5%,1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or smaller diameter or surface areaof the length, width or surface of the matrix.
 5. The test device ofclaim 1, wherein the distance between edges of at least two of thereagent dots is about 1-10 um, 10-50 um, 51-100 um, 101-200 um, 201-300um, 301-400, 401-500, or 501-600 um.
 6. The test device of claim 1,wherein at least a quarter, a third, half or all reagent dots comprise areagent that is capable of specifically binding to an analyte or anotherbinding reagent that is capable of binding to an analyte.
 7. The testdevice of claim 1, which is configured for a competitive test. 8-15.(canceled)
 16. The test device of claim 1 wherein the plurality ofreagent dots comprises the same binding reagent that is capable ofbinding to an analyte or another binding reagent that is capable ofbinding to the analyte, the plurality of reagent dots form multiplelines that are substantially parallel to the liquid sample flowdirection, the reagent dots in each line comprise the same amount of thebinding reagent, but the reagent dots in different lines comprise thedifferent amounts of the binding reagent, as the liquid sample flowslaterally alone the test device, the analyte, if present in the liquidsample, becomes sequentially bound to the binding reagent at each of thereagent dots in each of the lines until the analyte is depleted bybinding to the upstream reagent dot(s) in each of the lines, the bindingof the analyte to the reagent dot(s) generates a dateable signal at thereagent dot(s), and the intensity and/or the number of the dateablesignal at the reagent dot(s) provides a quantitation or asemi-quantitation of the analyte in the liquid sample.
 17. The testdevice of claim 16, wherein from one end to the other end of the testdevice, in the direction perpendicular to the direction of said liquidsample flow, the reagent dots in different lines comprise thesequentially different amounts of the binding reagent.
 18. The testdevice of claim 16, wherein the plurality of reagent dots comprises atleast 10, 50, 100, 500, 1,000, 5,000, 10,000 or more reagent dots. 19.The test device of claim 16, wherein at least a quarter, a third, halfor all reagent dots do not overlap and are sufficiently spaced apartfrom each other so that when the liquid sample flows laterally along thematrix, flow of the liquid sample to, through and/or around one of thereagent dots does not substantially affect flow of the liquid sample to,through and/or around the other reagent dots.
 20. The test device ofclaim 16, wherein at least a quarter, a third, half or all reagent dotshave a diameter of about 0.1-1 um, 1-10 um, 10-50 um, 51-100 um, 101-200um, 201-300 um, 301-400 um, 401-500 um or 501-1000 um, or at least aquarter, a third, half or all reagent dots have a diameter or surfacearea that is about 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001% orsmaller diameter or surface area of the length, width or surface of thematrix.
 21. The test device of claim 16, wherein the distance betweenedges of at least two of the reagent dots is about 1-10 um, 10-50 um,51-100 um, 101-200 um, 201-300 um, 301-400, 401-500, or 501-600 um. 22.The test device of claim 16, wherein at least a quarter, a third, halfor all reagent dots comprise a reagent that is capable of specificallybinding to an analyte or another binding reagent that is capable ofbinding to an analyte.
 23. The test device of claim 16, which isconfigured for a competitive test. 24-30. (canceled)
 31. The test deviceof claim 1, wherein the predetermined pattern is selected from the groupconsisting of a line, multiple lines, a symbol, a geometric shape and analpha-numeric shape.
 32. The test device of claim 31, wherein thealpha-numeric shape is a letter, a word, a number or a combinationthereof.
 33. The test device of claim 31, wherein the line issubstantially parallel to the liquid sample flow direction.
 34. The testdevice of claim 31, wherein the line is substantially perpendicular tothe liquid sample flow direction.
 35. The test device of claim 1,wherein at least a quarter, a third, half or all reagent dots havesubstantially the same size or diameter.
 36. The test device of claim 1,wherein at least one of the reagent dots has a shape selected from thegroup consisting of a line, a circle, a rod, a square, a triangle, arectangle and an irregular shape.
 37. The test device of claim 36,wherein at least a quarter, a third, half or all reagent dots have ashape selected from the group consisting of a line, a circle, a rod, asquare, a triangle, a rectangle and an irregular shape.
 38. The testdevice of claim 1, wherein at least a quarter, a third, half or allreagent dots have the same shape.
 39. The test device of claim 1,wherein the distance between at least a quarter, a third, half or allreagent dots is substantially the same.
 40. The test device of claim 1,which comprises multiple layers of the plurality of reagent dots. 41.The test device of claim 40, which comprises at least a layer of theplurality of reagent dots on both sides of the matrix.
 42. The testdevice of claim 1, wherein the matrix has a porous structure.
 43. Thetest device of claim 42, wherein the matrix comprises nitrocellulose,glass fiber, polypropylene, polyethylene, polyvinylidene flouride,ethylene vinylacetate, acrylonitrile and/or polytetrafluoro-ethylene.44. The test device of claim 1, wherein the matrix has a non-porousstructure.
 45. The test device of claim 1, wherein the matrix comprisesa plastics, a film of a matrix having a hydrophilic surface, or amaterial with a controlled contact angle with the sample liquid.